Phosphoenolpyruvate Provision to Plastids Is Essential for ...Phosphoenolpyruvate Provision to Plastids Is Essential for Gametophyte and Sporophyte Development in Arabidopsis thaliana

Phosphoenolpyruvate Provision to Plastids Is Essential forGametophyte and Sporophyte Development inArabidopsis thaliana C W

Veena Prabhakar,a Tanja Lottgert,a,1 Stefan Geimer,b Peter Dormann,c Stephan Kruger,a Vinod Vijayakumar,a

Lukas Schreiber,d Cornelia Gobel,e Kirstin Feussner,f Ivo Feussner,e Kay Marin,g Pia Staehr,a Kirsten Bell,a

Ulf-Ingo Flugge,a and Rainer E. Hauslera,2

a Universitat zu Koln, Biozentrum, Botanisches Institut II, D-50674 Cologne, Germanyb Universitat Bayreuth, Zellbiologie/Elektronenmikroskopie NW I/B1, D-95447 Bayreuth, Germanyc Universitat Bonn, Molekulare Biotechnologie, D-53115 Bonn, Germanyd Universitat Bonn, Institut fur Zellulare und Molekulare Botanik, Ecophysiology of Plants, D-53115 Bonn, GermanyeGeorg August University, Albrecht von Haller Institute for Plant Sciences, Ernst Caspari Building, Department of Plant

Biochemistry, D-37077 Goettingen, Germanyf Georg August University, Institute for Microbiology and Genetics, Department of Molecular Microbiology and Genetics,

D-37077 Goettingen, Germanyg Universitat zu Koln, Institut fur Biochemie, D-50674 Cologne, Germany

Restriction of phosphoenolpyruvate (PEP) supply to plastids causes lethality of female and male gametophytes in

Arabidopsis thaliana defective in both a phosphoenolpyruvate/phosphate translocator (PPT) of the inner envelope

membrane and the plastid-localized enolase (ENO1) involved in glycolytic PEP provision. Homozygous double mutants of

cue1 (defective in PPT1) and eno1 could not be obtained, and homozygous cue1 heterozygous eno1 mutants [cue1/eno1

(+/2)] exhibited retarded vegetative growth, disturbed flower development, and up to 80% seed abortion. The phenotypes of

diminished oil in seeds, reduced flavonoids and aromatic amino acids in flowers, compromised lignin biosynthesis in stems,

and aberrant exine formation in pollen indicate that cue1/eno1(+/2) disrupts multiple pathways. While diminished fatty acid

biosynthesis from PEP via plastidial pyruvate kinase appears to affect seed abortion, a restriction in the shikimate pathway

affects formation of sporopollonin in the tapetum and lignin in the stem. Vegetative parts of cue1/eno1(+/2) contained

increased free amino acids and jasmonic acid but had normal wax biosynthesis. ENO1 overexpression in cue1 rescued the

leaf and root phenotypes, restored photosynthetic capacity, and improved seed yield and oil contents. In chloroplasts,

ENO1 might be the only enzyme missing for a complete plastidic glycolysis.

INTRODUCTION

Phosphoenolpyruvate (PEP) plays a central role in plant metab-

olism. As an intermediate of glycolysis, PEP is indispensable for

energy metabolism in the cytosol and delivers ATP and pyruvate

by the action of cytosolic pyruvate kinase (PK) (Plaxton, 1996;

Givan, 1999). Pyruvate can be fed into the citric acid cycle,

yielding NADH for respiratory ATP generation (Fernie et al.,

2004). Inside the plastids, PEPmay act as a precursor for at least

four metabolic pathways (Figure 1A).

Together with erythrose 4-phosphate, PEP is fed into the

shikimate pathway, which delivers essential aromatic amino

acids and a large number of secondary plant products. The initial

steps of the shikimate pathway are exclusively localized within

the plastid stroma (Herrmann, 1995; Schmid and Amrhein, 1995;

Herrmann andWeaver, 1999). Inside the stroma, PEPcan also be

sequentially metabolized to pyruvate and acetyl-CoA by plastid

PK and the pyruvate dehydrogenase complex (Reid et al., 1977;

Elias and Givan, 1979; Lernmark and Gardestrom, 1994) and

thus enter the biosynthesis of fatty acids (Dennis, 1989;Ohlrogge

and Jaworski, 1997), which are quantitatively important for

triacylglycerol production in oil seeds (e.g., Voelker and Kinney,

2001; Rawsthorne, 2002; Ruuska et al., 2002). Like the shikimate

pathway, the de novo biosynthesis of fatty acids for membranes

and storage lipids is localized to the plastids (Ohlrogge et al.,

1979; Ohlrogge and Jaworski, 1997). Moreover, stromal pyru-

vate can act as a precursor for the synthesis of branched-chain

amino acids (Schulze-Siebert et al., 1984) and together with

glyceraldehyde 3-phosphate for the mevalonate-independent

way (2-C-methyl-D-erythritol 4-phosphate [MEP] pathway) of

isoprenoid biosynthesis (Lichtenthaler, 1999). Unlike plastids

1Current address: Quintiles GmbH, Hugenottenallee 167, D-63236 Neu-Isenburg, Germany.2 Address correspondence to [email protected] author responsible for distribution of materials integral to thefindings presented in this article in accordance with the policy describedin the Instructions for Authors (www.plantcell.org) is: Rainer E. Hausler([email protected]).CSome figures in this article are displayed in color online but in blackand white in the print edition.WOnline version contains Web-only data.www.plantcell.org/cgi/doi/10.1105/tpc.109.073171

The Plant Cell, Vol. 22: 2594–2617, August 2010, www.plantcell.org ã 2010 American Society of Plant Biologists

from photoautotrophic or mixotrophic tissues, such as green

oilseeds (Ruuska et al., 2004; Li et al., 2006; Schwender et al.,

2006), in plastids from nongreen tissues the above pathways rely

entirely on the provision of carbon skeletons and energy from the

cytosol.

In principle, fatty acid biosynthesis in nongreen plastids could

be driven by the import of glucose-6-phosphate (Glc6P) or triose

phosphates malate or pyruvate (Smith et al., 1992; Kang and

Rawsthorne, 1994; Qi et al., 1995; Eastmond and Rawsthorne,

2000; Ruuska et al., 2002; Figure 1A). In Brassicaceae, such as

canola (Brassica napus) or Arabidopsis thaliana, PEP is likely to

be the predominant precursor for fatty acid biosynthesis in seeds

(Schwender and Ohlrogge, 2002; Schwender et al., 2003; Andre

et al., 2007, Baud et al., 2007b; Lonien and Schwender, 2009).

Hence, a sufficient provision of PEP appears to be essential for

lipid biosynthesis and storage (Kubis et al., 2004). In principle,

pyruvate generated by cytosolic PK may be imported as pre-

cursor for fatty acid biosynthesis, which is supported by feeding

experiments with 14C-labeled pyruvate to isolated plastids from

B. napus embryos and the subsequent incorporation of 14C into

fatty acids (Kang and Rawsthorne, 1994; Eastmond and

Rawsthorne, 2000). However, pyruvate deriving from stromal

PEP serves as the major precursor for fatty acid biosynthesis in

plastids of developing oil seeds (Figure 1A). This notion is sup-

ported by the observation that a restriction in plastid-localized

PK, which converts PEP to pyruvate, resulted in severely dimin-

ished seed oil contents (Andre et al., 2007; Baud et al., 2007b)

and by recent 13C feeding experiments of Arabidopsis embryos

(Lonien and Schwender, 2009).

PEP can be delivered by the phosphoenolpyruvate/phosphate

translocator (PPT) from the cytosol (Fischer et al., 1997) or may

be generated inside the plastids by a complete glycolytic path-

way (Figure 1A). However, chloroplasts and most nongreen

plastids lack the ability to form PEP via glycolysis because the

essential enzymes, which convert 3-phosphoglycerate (3-PGA)

to PEP (i.e., phosphoglyceromutase [PGyM] and enolase [ENO])

are either absent or, if present, show a low activity (Stitt and Ap

Rees, 1979; Schulze-Siebert et al., 1984; Journet and Douce,

1985; Bagge and Larsson, 1986; Van der Straeten et al., 1991;

Miernyk and Dennis, 1992; Borchert et al., 1993). By contrast,

plastids from lipid storing tissues such as seeds of castor bean

(Ricinus communis) or canola have been demonstrated to be

capable of catalyzing glycolytic PEP formation (Eastmond and

Rawsthorne, 2000).

The import of PEP from the cytosol into the plastid stroma is

catalyzed by PPT. The genome of Arabidopsis contains two

PPT genes (PPT1, At5g33320; and PPT2, At3g01550), which

have been characterized at molecular and functional levels

(Knappe et al., 2003). PPT1 is defective in the chlorophyll a/b

binding protein underexpressed1 (cue1) mutant (Li et al., 1995;

Streatfield et al., 1999), which exhibits a reticulate leaf phenotype

with wild-type-like bundle sheath cells but aberrant mesophyll

cells and smaller chloroplasts therein (Kinsman and Pyke, 1998).

The involvement of a PPT in the delivery of PEP to plastids for the

production of aromatic amino acids in certain cell types has

already been demonstrated (Streatfield et al., 1999; Voll et al.,

2003). The cue1 mutant phenotype could be rescued by feeding

aromatic amino acids via the roots (Streatfield et al., 1999) or by

Figure 1. Metabolic Role of PEP in Plastids of Heterotrophic or Mixo-

trophic Tissues (i.e., Developing Seeds).

In wild-type plants (A), PEP can be imported from the cytosol by PPT, or

it may be produced from 3-PGA by the glycolytic sequence involving

PGyM and ENO. Both enzymes exist as plastidic and cytosolic forms. In

the stroma, PEP together with erythrose 4-phosphate (E-4-P) can enter

the shikimate pathway for the biosynthesis of aromatic amino acids and

derived compounds, or after conversion to pyruvate by PK, it can be fed

into the biosynthesis of fatty acids, isoprenoids, or branched-chain

amino acids. Pyruvate may also be imported by a pyruvate transporter

(PyT). Other transporters of the phosphate translocator family, such as

GPT or the triose phosphate/PT (TPT), may import Glc6P or 3-PGA,

respectively. Glc6P can be fed into OPPP and starch biosynthesis. Note

that TPT is not likely to be expressed in heterotrophic tissues. The OPPP

produces reducing equivalents in the form of NADPH required for

anabolic reactions and metabolic intermediates, such as E-4-P. In

mixotrophic plastids, 3-PGA and reducing equivalents can be produced

by the Calvin cycle (reductive pentose phosphate pathway [RPPP]). By

cytosolic glycolysis, imported sucrose can be metabolized to pyruvate,

which is subjected to respiration in the mitochondria. In (B), the conse-

quences of a deficiency in both PPT1 and ENO1 are shown. Most likely

all metabolic pathways shaded in light gray within the plastids would be

negatively affected, which would also feed back on processes taking

place in the cytosol.

Phosphoenolpyruvate in Arabidopsis Plastids 2595

the ectopic expression of a C4-type plastid-targeted pyruvate,

orthophosphate dikinase (PPDK), capable of producing PEP

from pyruvate (Voll et al., 2003), indicating that a shortage of PEP

in certain plastids of cue1 is responsible for the mutant pheno-

type. Moreover, the latter experiment indirectly supports either

the presence of a plastid pyruvate transporter in those cell or

tissue types where PPT1 is absent, a sufficient rate of pyruvate

diffusion across the envelope, or the activity of a plastid-localized

malic enzyme, which is capable of producing pyruvate by oxi-

dative decarboxylation of malate (Wheeler et al., 2005). Pyruvate

import or generation within the plastid, linked to the activity of

PPDK, which is targeted to the cytosol and the plastids (Parsley

and Hibberd, 2006), might also be capable of providing PEP for

the shikimate pathway in certain tissues. However, the expres-

sion level of the Arabidopsis PPDK (At4g15530) based on micro-

array data is rather poor in most vegetative tissues, but it is

enhanced in mature pollen and imbibed seeds (http://bar.

utoronto.ca/efp/cgi-bin/efpWeb.cgi; Winter et al., 2007). It is

hence more likely that, in addition to the import from the cytosol

by a PPT, PEP is generated by a complete glycolytic pathway in

the plastids.

We have recently identified and functionally characterized the

plastid-localized enolase (ENO1) of Arabidopsis, which catalyzes

the glycolytic conversion of 2-phosphoglycerate (2-PGA) to PEP

(Prabhakar et al., 2009). Most strikingly, the single-copy gene

ENO1 is not expressed in photosynthetic tissues but exhibits high

transcript abundance indeveloping siliques.Moreover, bothENO1

and PPT1 are coexpressed during early embryo and seed devel-

opment (http://bar.utoronto.ca/efp/cgi-bin/efpWeb.cgi), suggest-

ing that the task of PEP provision to plastids in these tissuesmight

be shared both by import from the cytosol and by a complete

glycolytic pathway within the stroma (Prabhakar et al., 2009).

Here, we demonstrate that Arabidopsis double mutant plants

defective in both PPT1 and ENO1 are not viable due to partial

gametophytic lethality and impaired sporophyte development,

underlining the importance of plastidic PEP (Figure 1B). Ectopic

overexpression of ENO1 under the control of the cauliflower

mosaic virus (CaMV) 35S promoter could rescue the phenotype

of the cue1mutant. The role of PEP in plastids of generative and

vegetative tissues of Arabidopsis is discussed.

RESULTS

A Double Knockout of PPT1 and ENO1 Is Lethal

PPT1 and ENO1 are coexpressed during early embryo and seed

development and most likely share the provision of PEP to

plastids for anabolic reaction sequences during this develop-

mental stage (Prabhakar et al., 2009). To elucidate the conse-

quences of a restriction in PEP supply to plastids, the eno1-1

mutant allele (Prabhakar et al., 2009) was crossed with the

cue1-1mutant allele (Li et al., 1995), which represents a deletion

of the PPT1 gene locus (Streatfield et al., 1999) and likely also at

least five expressed genes adjacent to PPT1 (R.E. Hausler and

U.-I. Flugge, unpublished data). Among the F2 generation, 300

plants homozygous for cue1-1 were analyzed for the T-DNA

insertion in the ENO1 gene. In this screen, no double homozy-

gousmutants and only four eno1 heterozygous plants (i.e., 1.3%)

were found (Table 1). This low percentage was far below 50%

expected. Moreover, heterozygous eno1-1 mutants in the ho-

mozygous cue1-1 background [cue1-1/eno1-1(+/2), ccEe] ex-

hibited stunted shoot growth and retarded flowers and siliques.

In the progeny of these ccEe plants (F3 generation), again no

double homozygous mutants (ccee) and only four heterozygous

mutants (ccEe), all with a retarded growth phenotype, were

found among 74 tested plants. This observation suggests that a

combined mutation in both genes results in lethality. To confirm

this assumption and to further elucidate the observed segrega-

tion pattern, crosses between a secondmutant allele defective in

ENO1 (eno1-2; Prabhakar et al., 2009) and three different cue1

alleles (cue1-1, cue1-3, and cue1-6) were generated. Like cue1-1,

cue1-3 is in the background of the ecotype Bensheim (i.e.,

pOCA106; Li et al., 1995) and is described as a weak allele of

cue1, whereas cue1-6 represents a strong allele in theColumbia-0

(Col-0) background (Streatfield et al., 1999). Both cue1-3 and

cue1-6were obtained from ethyl methanesulfonate–mutagenized

plants. Analogous to the crosses of eno1-1with cue1-1, no double

homozygous mutants could be isolated in the additional crosses,

and the percentage of heterozygous eno1 mutants in the homo-

zygous cue1backgroundswas again far below expectation (Table

1). These data indicate that lethality of the double homozygous

cue1/eno1mutants is due to a defect in both the PPT1 and ENO1

genes and is not caused by any secondary T-DNA insertions or

mutations in the single mutant backgrounds as is evident for

cue1-1.

A Heterozygous Mutation of ENO1 in the Background of

cue1 Leads to Stunted Shoot Growth and Aberrant

Flower Development

Plants heterozygous for eno1 in the homozygous cue1 back-

ground (ccEe) exhibited a stunted growth phenotype compared

with the wild type or the cue1 single mutants (Figure 2A).

Interestingly, growth retardation became evident only after the

plants were grown for 4 to 5 weeks on soil (see Supplemental

Figure 1A online), whereas plants grown on Murashige and

Skoog (MS) agar plates for up to 3 weeks lack any visible growth

phenotype of the roots or the shoot (see Supplemental Figure 1B

online). Besides the retarded growth of the shoot, the develop-

ment of the first flowers was severely hampered and resulted in

Table 1. Genetic Analyses of Crosses between cue1 (Male) and eno1

(Female) Mutants

Mutant Cross ccEe Plants (%)

Homozygous cue1

Mutants Analyzed in

Each Harvest

cue1-1 3 eno1-1 3.79 6 0.18 66 to 71

cue1-1 3 eno1-2 1.43 6 0.01 68 to 73

cue1-3 3 eno1-2 16.38 6 0.22 53 to 57

cue1-6 3 eno1-2 13.08 6 0.09 56 to 65

The frequency of heterozygous eno1 mutants in the homozygous cue1

background (ccEe plants) is shown as a percentage of plants analyzed

in three independent harvests (mean 6 SE).

2596 The Plant Cell

Figure 2. Phenotype of Heterozygous eno1 Mutants in the Homozygous cue1 Background (ccEe) Compared with Wild-Type and cue1 Plants Grown

for 8 Weeks in the Greenhouse.

(A) Comparison of the growth phenotype of Col-0 (1), eno1-2 (2), cue1-1 (3), and cue1-1/eno1-2 (4) plants. The inset shows a detailed view of cue1-1/

eno1-2(+/�) (5).

(B) Opened bud of an early (stage 10) wild-type (Col-0) flower.

(C) Opened bud of an early (stage 10) flower of cue1-1/eno1-2(+/�) with degenerated stamens.

(D) Flower and silique development of cue1-6.

(E) Schematic representation of the position of flowers and siliques shown in (D) and (F)

(F) Flower and silique development of cue1-6/eno1-2(+/�) plants.

(G) Destained mature siliques of Col-0 (1), pOCA (2), cue1-1 (3), cue1-6/eno1-2(+/�) (4), and cue1-1/eno1-2(+/�) (5)

Developmental stages of flowers and siliques shown in (D) and (F) are based on the position of the flowers/siliques at the raceme starting from the

topmost to lower positions.

The numbers in (E) represent the positions of flowers and/or siliques shown in (D) and (F). Bars = 5 mm in (D) and (F).


deformed and infertile flowers with underdeveloped stamen

(Figures 2B and 2C). At the later stages, flower development

was less affected and siliques were formed after pollination

(Figures 2D to 2F). However, the size of the siliques was dimin-

ished and their length was reduced by >50% compared with

wild-type plants or the singlemutants (Figure 2G). The number of

seeds per silique was severely diminished in all ccEe plants

from ;50 seeds in Col-0 or pOCA to <10% in cue1-1/eno1-2

(+/2) and between 30 and 40% in cue1-3/eno1-2(+/2) and

cue1-6/eno1-2(+/2), respectively (Table 2). There was also a

decline in the seed number per silique in the cue1 single mutant

alleles, which was less marked for the cue1-3 or cue1-6 allele.

According to the gaps in the mature siliques of ccEe plants

(Figure 2G), an abortion frequency of seeds of >80% was

calculated for the progeny of cue1-1 3 eno1-2 crosses and

;45% for both crosses of cue1-33 eno1-2 and cue1-63 eno1-2

(Table 2). The cause for the developmental constraints in ccEe

plants was further elucidated by expression studies of ENO1. We

could show previously that ENO1 is highly expressed in nonroot

hair cells of the roots and in the shoot apex but is absent in

mature leaves (Prabhakar et al., 2009). As revealed by quantita-

tive RT-PCR (qRT-PCR), ENO1 expression in the roots and the

shoot apex of the developing rosettes of cue1-6/eno1-2(+/2)

was diminished by 29% 6 4% (n = 3) and 69% 6 6% (n = 3),

respectively, compared with the wild type. These data indicate

that a heterozygous knockout mutation of ENO1 results in a

substantial decrease in ENO1 expression and suggest that the

growth retardation in the ccEe lines is based on a gene dosage

effect.

Female and Male Gametophyte Development and Seed

Formation Are Impaired in cue1/eno1(+/2) Plants

To address the question whether or not gametophyte develop-

ment was hampered in ccEe, we further analyzed the pheno-

types of ovules in flower buds at stage 10 (Smyth et al., 1990)

before pollination (Figure 3). Besides ovules with a wild-type-like

appearance and a normally developed embryo sac (Figures 3A

and 3B), in;40% of the ovules the embryo sac was diminished

in size (Figure 3C) or even absent (Figure 3D). Moreover, there

were also ovules completely halted in development (Figure 3E).

The above data show that a combined knockout of PPT1 and

ENO1 can result in lethality of the female gametophyte.

The remaining developed and matured seeds resulting from

unaffected ovules after fertilization could be categorized into

three different classes according to their size and phenotypic

appearance. Class I seeds were indistinguishable from seeds

of cue1 (Figure 3G), which are slightly lighter in color compared

with wild-type seeds and hence resemble those from transpar-

ent testa mutants (Koornneef, 1990) (Figure 3F). By contrast,

class II and class III seeds were intermediately and severely

diminished in size, respectively (Figure 3H). Moreover, class III

seeds exhibited a wrinkled and shrunken appearance with

irregularly shaped testa cells similar to the wrinkled1 (wri1)

mutant (Focks and Benning, 1998; Cernac et al., 2006; Baud

et al., 2007a). Table 2 shows the distribution of the individual

seed classes between the different plant lines. There was also a

higher percentage of class II and III seeds in the eno1 single

mutant alleles as well as cue1-1 compared with the wild type. A

genotypic analysis of all three seed classes revealed that class I

seeds were wild-type for ENO1, whereas both class II and class

III seeds were heterozygous for eno1. Class I and II seeds were

capable of germinating on MS agar plates, whereas class III

seeds were not.

We further analyzed the effect of a double knockout of PPT1

and ENO1 on the development of the male gametophyte. Cross

sections of anthers of ccEe plants revealed a reduced number of

pollen compared with the wild type (Figures 4A to 4C). Moreover,

there was a large portion of underdeveloped pollen in anthers of

cue1-1/eno1-2(+/2) and cue1-6/eno1-2(+/2) plants (Figures 4B

Table 2. Frequencies of Seed Abortion, Seed Phenotypes, and Pollen Viability in Wild-Type Arabidopsis (Col-0, Bemsheim [pOCA]), eno1 and cue1

Alleles, and Heterozygous eno1 Mutants in the Homozygous cue1 Background (ccEe Plants)

Plant Line

Seed Abortion

Seed Phenotype (%)

Nonviable Pollen (%)

Number of Seeds

per Silique Number of Gaps Abortion (%) Class I Class II Class III

Col-0 50.3 6 2.2 0.25 6 0.13 0.5 (n = 12) 97.70 2.30 0.00 0.3 6 0.3

pOCA 47.9 6 1.1 1.00 6 0.29 2.0 (n = 9) 95.00 5.00 0.00 0.3 6 0.3

eno1-1 48.9 6 0.8 1.44 6 0.48d 2.9 (n = 9) 80.00 17.00 3.00 6.3 6 2.0a

eno1-2 43.8 6 0.8c 1.00 6 0.37 2.2 (n = 9) 76.00 20.00 4.00 8.0 6 1.0a

cue1-1 32.9 6 1.1a 8.89 6 0.72a 21.3 (n = 9) 70.00 25.00 5.00 0.6 6 0.3

cue1-3 45.3 6 2.0 5.44 6 0.84a 10.7 (n = 9) 91.10 8.90 0.00 0.3 6 0.3

cue1-6 41.0 6 2.2b 1.30 6 0.35c 3.1 (n = 6) 91.10 8.90 0.00 0.3 6 0.3

cue1-1/eno1-2(+/�) 2.4 6 0.6a 12.40 6 0.65a 83.6 (n = 9) 65.40 25.00 9.60 35.0 6 2.7a

cue1-3/eno1-2(+/�) 20.2 6 2.3a 16.50 6 0.89a 45.0 (n = 27) 86.20 10.30 3.50 2.0 6 0.5

cue1-6/eno1-2(+/�) 16.2 6 2.3a 13.20 6 1.20a 44.9 (n = 26) 64.00 26.00 10.00 29.0 6 2.0a

Seeds and seed gaps were counted in 6 to 27 siliques per line (n). The developed seeds were categorized into three classes according to their

phenotypic appearance. Class I seeds were wild-type like, class II seeds showed an intermediate reduction in size, and class III seeds a strong

reduction in size. Pollen viability was tested by Alexander stain of pollen collected from three plants per line. The data are expressed as mean 6 SE.

Statistical significance of differences between the parameters were assessed by the Welch test with probability values of P < 0.001 (a), P < 0.01 (b),

P < 0.02 (c), and P < 0.05 (d).

2598 The Plant Cell

and 4C). Vitality staining showed that the underdeveloped pollen

grains of theccEeplantsweredead (Figures 4Dand4E). Likewise

the cellular structure of the underdeveloped pollenwas disrupted

and the DNA of the nuclei in the three cellular stage was not

detectable by 4’,6-diamidino-2-phenylindole (DAPI) staining

(Figures 4F to 4I). In the cue1-1/eno1-2(+/2) and cue1-6/eno1-

2(+/2) double mutants, the lethality frequency of the pollen was

up to 35 and 29%, respectively, compared with <1% in wild-

type plants and the single mutants. Interestingly, ccEe plants

obtained from crosses of eno1-2 with the weak cue1-3 allele

showed only 2% dead pollen, suggesting that at least pollen

viability can bemaintained if PPT1 is present, albeit functionally

impaired (Table 2).

Figure 3. Phenotypes of Ovules and Mature Seeds of Heterozygous

eno1 Mutants in the Homozygous cue1 Background (ccEe) Compared

with Wild-Type or cue1 Plants.

(A) Ovule of a wild-type (Col-0) plant.

(B) Ovule of cue1-1/eno1-2(+/�) with a wild-type-like appearance.

(C) Ovule of cue1-1/eno1-2(+/�) with a swollen embryo sac.

(D) Ovule of cue1-1/eno1-2(+/�) lacking an embryo sac.

(E) Degenerated ovule of cue1-1/eno1-2(+/�).

(F) Wild-type (pOCA) seeds.

(G) Seeds of cue1-1.

(H) Phenotype of seeds of cue1-1/eno1-2(+/�) showing either a wild-

type-like appearance (class I seeds; 1) or that were intermediately and

strongly reduced in size (class II, 2; and class III seeds, 3).

Bar = 50 mm in (A) to (E).

[See online article for color version of this figure.]

Figure 4. Cross Sections of Pollen Sacs as well as Vitality (Alexander)

and DAPI Staining of Pollen from Heterozygous eno1 Mutants in the

Homozygous cue1 Background (ccEe) Compared with the Wild Type.

(A) Cross section of a wild-type (Col-0) pollen sac.

(B) Cross section of a pollen sac of cue1-1/eno1-2(+/�).

(C) Cross section of a pollen sac from cue1-6/eno1-2(+/�).

(D) Vitality staining of wild-type pollen (pOCA).

(E) Vitality staining of pollen from cue1-1/eno1-2(+/�)with an example of

a viable (1) and an aborted (2) pollen.

(F) Bright-field image of wild-type pollen (pOCA).

(G) DAPI staining of the wild-type pollen shown in (F).

(H) Bright-field image of wild-type-like pollen and degenerated pollen

(arrow) of cue1-1/eno1-2(+/�).

(I) DAPI staining of the pollen grains shown in (H). The degenerated

pollen is marked by an arrow.

[See online article for color version of this figure.]


The Pollen Exine Structure Is Impaired and Phenolic

Compounds Are Less Abundant in Anthers and Mature

Pollen of ccEe Plants

To further elucidate the reason for the high lethality rate of ccEc

pollen, we analyzed the ultrastructure of developing pollen grains

of wild-type and ccEe plants. As shown in Figure 5, pollen grains

from ccEe plants exhibit a variety of phenotypes different from

wild-type pollen (Figures 5A and 5B). The most prominent

features of ccEe compared with wild-type pollen grains were

an irregularly shaped intine (Figures 5D, 5F, 5H, and 5L) and a

strongly diminished exine structure (Figures 5F to 5J). In individ-

ual cases, pollen development was completely disrupted (Fig-

ures 5K and 5L).Moreover, in someof themutant pollen,massive

starch accumulation was found in the plastids (Figures 5D, 5G,

and 5H), whereas starch granules in wild-type pollen were less

abundant (Figures 5A and 5B). Interestingly, lipid bodies and

vacuoles were found at nearly the same density in ccEe pollen

grains (Figures 5C to 5J) compared with the wild type (Figures 5A

and 5B), suggesting that fatty acid biosynthesis appears not to

be a major metabolic restriction during pollen maturation. Apart

from dead pollen (Figures 5K and 5L), deposition of exine

material was severely diminished in pollen of ccEe plants (Fig-

ures 5C to 5L), suggesting that sporopollonin deposition was

restricted. Sporopollonin represents the major constituent of the

exine and consists of both long-chain fatty acids and phenolic

compounds derived from the phenylpropanoid metabolism

(Guilford et al., 1988; Wiermann et al., 2001; Dobritsa et al.,

Figure 5. Cross Sections of Differently Affected Pollen Grains of Heterozygous eno1Mutants in the Homozygous cue1 Background (ccEe) Analyzed by

Transmission Electron Microscopy in the Tricellular Stage in Comparison to Wild-Type Col-0.

(A) Cross section of a wild-type (Col-0) pollen grain.

(B) Close-up of the wild-type pollen grain shown in (A).

(C) to (L) Phenotypic changes in the ultrastructure of pollen grains observed in pollen sacs of ccEe plants.

(C) Pollen grain of cue1-1/eno1-2(+/�) with a wild-type-like appearance.

(D) Close-up of the pollen grain shown in (C) with increased numbers of starch granules in the plastids and a slightly deformed and swollen intine.

(E) Pollen grain of cue1-1/eno1-2(+/�) with a wild-type-like size but affected exine and intine structures.

(F) Close-up of the pollen grain shown in (E) with a focus on the underdeveloped exine structure and the strongly deformed intine.

(G) Pollen grain of cue1-1/eno1-2(+/�) with a deformed pollen wall, high numbers of starch granules, and large vacuole-like structures.

(H) Close-up of the pollen grain shown in (G) with a focus on the impaired exine and intine structures.

(I) Pollen grain of cue1-6/eno1-2(+/�) with a wild-type-like appearance but an impaired exine structure.

(J) Close-up of the pollen grain shown in (I) with a focus on the underdeveloped exine structure.

(K) Strongly deformed pollen grain of cue1-6/eno1-2(+/�).

(L) Close-up of the pollen grain shown in (K).

Ex, exine; In, intine; L, lipid body; ER, endoplasmatic reticulum; V, vacuoles; VL, vacuole-like bodies; M, mitochondria; P, plastids; S, starch granules.

Bars = 2 and 0.5 mm for the overviews and close-ups, respectively.

2600 The Plant Cell

2009). Moreover, a comparison of autofluorescence emission

from developing anthers captured by confocal laser scanning

microscopy revealed that phenolic compounds were less abun-

dant both in the anthers and in the pollen exine of ccEe plants

compared with the wild type (see Supplemental Figure 2 online).

Likewise, diphenylborate-2-aminoethyl–stained mature pollen

grains of ccEe plants exhibited considerably diminished yellow/

green fluorescence both from the wall and the cytoplasm of the

pollen grains compared with the wild type (see Supplemental

Figure 3 online).

In Vitro Germination of Pollen Is Severely Diminished in

ccEe Plants

The developmental constrains of pollen from ccEe plants com-

bined with their aberrant ultrastructure and diminished contents of

phenolic compounds are reflected in a severely diminished in vitro

germination rate. As is shown in Supplemental Figure 4 online,

<20% of the pollen grains were capable of germinating in the lines

cue1-1/eno1-2(+/2) and cue1-6/eno1-2(+/2), whereas almost

80% of pollen from wild-type plants germinated on agar plates.

Male and Female Transmission Efficiencies of the cue1 and

eno1MutationWere Diminished in Segregating ccEe Plants

Having established that a high lethality frequency of both the

female and male gametophytes in ccEe plants was involved in

the failure to establish double homozygous cue1/eno1 mutant

plants, we further analyzed the segregation pattern of the indi-

vidual genotypes in the F2 generation of crosses between cue1-1

or cue1-6 and eno1-2. To determine female and male transmis-

sion efficiencies (TEs), reciprocal crosses between eno1-2 and

cue1-1 were performed. A detailed analysis of the segregation

patterns is contained in Supplemental Table 1 online. The major

outcome of this analysis can be summarized as follows. For the

mutation in the PPT1 gene, both female and male TEs were

similar (30%) but lower than the expected value of 50% for each

gametophyte. By contrast, the female TE for the mutation in the

ENO1 gene was about half (16.7%) compared with the male TE

(31.3%), suggesting that a lesion of ENO1 in the background of

the homozygous cue1 mutant has a much stronger effect on

female gametophyte development (in particular on embryo sac

development) than a lesion in the PPT1 gene in the homozygous

eno1 background.

Contents of Aromatic andBranched-ChainAminoAcidsAre

Diminished in Flowers and Rosette Leaves of cue1/eno1

(+/2) Double Mutants

The proposed function of the PPT in vegetative tissues is the

provision of PEP for the shikimate pathway (Fischer et al., 1997),

and it has previously been shown that a mutation in the PPT1

gene in cue1 mutants leads to lower contents of Phe in leaves

(Voll et al., 2003). Moreover, branched-chain amino acid synthe-

sis commences from plastidic pyruvate via acetolactate syn-

thase (Schulze-Siebert et al., 1984). To gain information on the

steady state contents of amino acids in those tissues where both

PPT1 and ENO1 are expressed compared with tissues where

ENO1 is absent, we analyzed the amino acid composition in

mature flowers (stage 13-14; Smyth et al., 1990) and rosette

leaves of ccEe plants and the single mutants compared with

wild-type plants (Figure 6; see Supplemental Figure 5 online).

In flowers, the content of total free amino acids (i.e., the sum of

all detected amino acids after HPLC separation) ranged between

3 and 4 mmol·g21 fresh weight (FW) and was not significantly

affected in the individual mutant lines compared with wild-type

plants (Figure 6A). Of the aromatic amino acids, Phe contents

varied between the lines but did not show a significant change in

either of themutants compared with wild-type plants (Figure 6B).

By contrast, Tyr content was significantly increased in both

alleles of the eno1 mutant but only slightly diminished in cue1-6

aswell as in the ccEeplants (Figure 6C). Similarly, the Trp content

was increased in both eno1 mutant alleles but decreased in the

ccEe plants (Figure 6D). Of the branched-chain amino acids, the

contents of Val and Leu were not significantly altered between

the lines (Figures 6E and 6F), whereas Ile content was diminished

in cue1-6 and the ccEe plants (Figure 6G). In Supplemental

Figure 3 online, the contents of a broader range of proteogenic

amino acids are shown for the mutant compared with wild-type

plants. Of the major amino acids, Gln content was significantly

diminished by ;50% in both eno1 mutant alleles and the ccEe

plants (see Supplemental Figure 5B online), whereas Glu content

was increased in cue1-6 but not consistently affected in both

ccEe alleles (see Supplemental Figure 5A online). Thr and Gly

contents showed a trend of an increase in both eno1 alleles but

were diminished in cue1-6 (see Supplemental Figures 5E and 5F

online), whereas Ser content remained unaffected (see Supple-

mental Figure 5D online). The content of Ala was increased in all

mutant lines compared with the wild-type plants (see Supple-

mental Figure 5G online). His content was significantly dimin-

ished by 30% in the ccEe plants (see Supplemental Figure 5H

online). It has recently been shown that His exerts control on

developmental processes (Mo et al., 2006). Interestingly, both

Arg and Lys contents were significantly increased in cue1-6 but

exhibited wild-type-like levels in both eno1 alleles and the ccEe

plants (see Supplemental Figures 5I and 5J online). An increased

Arg content in cue1 has previously been reported by Streatfield

et al. (1999) and He et al. (2004). For the latter report, a new cue1

mutant allele (nos1) has been isolated in a screen for nitric oxide

(NO) overproducers.

A different picture emerged from the amino acid spectrum of

mature rosette leaves (i.e., where ENO1 is not expressed). The

total amino acid content was doubled in cue1-6 and increased

even threefold in cue1-6/eno1-2(+/2) compared with the wild

type and the eno1-2mutant (Figure 6H). This increase in the total

amino acid content reflects specifically higher contents of Glu,

Gln, Asp, Asn, Ser, and Thr. When expressed as a percentage of

the total free amino acids, the content of the major amino acids

remained nearly unaltered between the plants (seeSupplemental

Figures 5K to 5O online). By contrast, the relative content of Gly

was diminished in cue1-6 and cue1-6/eno1-2(+/2) (see Supple-

mental Figure 5P online), whereas Alawas significantly increased

in both these lines (see Supplemental Figure 5Q online). The

relative contents of minor amino acids, such as the aromatic

amino acids Phe, Tyr, and Trp (Figures 6I to 6K), as well as the

branched chain amino acids Val, Leu, and Ile (Figures 6L to 6N)


Figure 6. Contents of Selected Amino Acids Extracted from Flower Buds or Rosette Leaves of the Wild Type (Col-0), cue1-6 and eno1 Single Mutant,

and the Heterozygous eno1 Mutants in the Homozygous cue1 Background (ccEe).

Flower buds ([A] to [G]) and rosette leaves ([H] to [N]). The data represent the mean 6 SE of n = 5 ([A] to [G]) or n = 3 ([H] to [N]) independent

experiments. Statistical significance of differences between the parameters were assessed by theWelch test with probability values of P < 0.001 (a), P <

0.01 (b), and P < 0.05 (c) indicated above the respective bars. Contents of total soluble amino acid ([A] and [H]) were estimated from the sum of all

recognized proteinogenic amino acid after separation by HPLC. The relative contents of the aromatic amino acids Phe, Tyr, Trp ([B] to [D] and [I] to [K])

and the branched-chain amino acids Val, Leu, and Ile ([E] to [G] and [L] to [N]) were expressed as a percentage fraction of the total amino acid content

in flower buds (A) and rosette leaves (H), respectively.

2602 The Plant Cell

were significantly decreased on the expense of the major amino

acids in cue1 and in particular in ccEe plants. However, on an

absolute scale, only Phe was lowered in cue1-6/eno1-2(+/2),

whereas Tyr content remained unaltered and Trp content was

even slightly enhanced. In contrast with flowers, relative and

absolute contents of His and Arg were significantly increased in

rosette leaves of cue1-6/eno1-2(+/2) (see Supplemental Figures

5R and 5S online).

Phytohormone Levels Are Altered in Flowers and Rosette

Leaves of ccEe Plants

To gain additional information for the underlying reason of the

aberrant growth and developmental phenotype in ccEe plants,

we analyzed the levels of phytohormones and signal molecules

in the wild type, the cue1-6 and eno1-2 single mutants, as well

as in cue1-6/eno1-2(+/2) (Figure 7). The growth regulator

indole-3-acetic acid (IAA) has been of particular interest, as it

can derive from the aromatic amino acid Trp (Bartel, 1997). In

flowers, the IAA content seemed to be decreased in cue1-6 and

cue1-6/eno1-2(+/2) (Figure 7A), and in rosette leaves, it was

significantly increased (i.e., doubled) only in eno1-2 (Figure 7G).

Cytokinin levels were below detection limits with the method

applied. However, with the aid of an ultra performance liquid

chromatography/time-of-flight mass spectrometry (TOF-MS)

analysis of relative amounts of cis/trans zeatin could be

detected, and a twofold increase in rosette leaves of cue1-6

and cue1-6/eno1-2(+/2) was found. The content of abscisic

acid (ABA) was significantly increased in flowers but not in

rosette leaves of ccEe plants (Figures 7B and 7H). Interestingly,

in rosette leaves, the ABA level was significantly increased in

leaves of the eno1-2 single mutant (Figure 7H). Strikingly, levels

of jasmonic acid (JA) and its precursor 12-oxo-phytodienoic

acid (oPDA) were significantly increased in rosette leaves of

ccEe plants (Figures 7I and 7J) but remained unaltered in

flowers (Figures 7C and 7D). High levels of JA have been shown

to inhibit mitosis and thereby plant growth (Zhang and Turner,

2008). Salicylic acid (SA) responded with a decline in flowers

and an increase in rosette leaves only in the eno1-2 single

mutant (Figures 7E and 7K). The content of the SA glucoside

(SAG) remained unaltered in all lines both in flowers and in

rosette leaves (Figures 7F and 7L).

Figure 7. Contents of Phytohormones in Flowers or Rosette Leaves of Col-0 Wild Type, cue1-6 and eno1-2 Single Mutants, and the Heterozygous

eno1-2 Mutant in the Homozygous cue1-6 Background (cue1-6/eno1-2[+/�]).

Flowers ([A] to [F]) and rosette leaves ([G] to [L]). Phytohormones identified by liquid chromatography–mass spectrometry separation were IAA ([A] and

[G]), ABA ([B] and [H]), oPDA ([C] and [I]), JA ([D] and [J]), SA ([E] and [K]), and SAG ([F] and [L]). The data represent the mean 6 SE of n = 3

independent experiments. Statistical significance of differences between the parameters was assessed by the Welch test with probability values of

P < 0.01 (a), P < 0.02 (b), and P < 0.05 (c) indicated above the respective bars.


Secondary Plant Products Are Diminished in ccEe Plants

A variety of secondary plant products derive from phenylpropa-

noid metabolism, such as flavonoids (Dinkova-Kostova, 2008),

anthocyanidin (Chalker-Scott, 1999), and lignin (Boerjan et al.,

2003). Phenylpropanoids are synthesized from the aromatic amino

acid Phe as one of the end products of the shikimate pathway.

Flavonoids play a key role in UV protection (Dinkova-Kostova,

2008) or as putative signal molecules (e.g., Buer et al., 2007). In

some species, flavonoids are also constituents of the cuticle or of

suberin layers (Holloway, 1983; Mintz-Oron et al., 2008). It is

conceivable that a decreased flux into aromatic amino acids and

derived compounds (such as flavonoids) leads to disturbed de-

velopment of the flowers aswas observed for the ccEe plants (see

Figure 2).

Flavonoid contents in mature flowers were quite variable be-

tween the lines and ranged between 6.51 and 9.64 nmol·g21 FW

(referred to naringinin as a flavonoid standard) in the control plant

pOCA and in Col-0 wild type, respectively. There was no clear

trend of diminished flavonoid content in the cue1 or eno1 single

mutants compared with the wild type (see Supplemental Table 2

online). By contrast, flavonoid contents in cue1-1 appeared to be

increased compared with the control plant. The flavonoid content

in the ccEe plant [cue1-6/eno1-2(+/2)] of 5.56 nmol·g21 FW was

significantly diminished by 42% compared with the wild type but

only 10% compared with the cue1-6 single mutant. Contents of

anthocyaninswere close to the detection limit in the flower budsof

all lines investigated and therefore are not shown.

Lignin, as a constituent of cell walls (e.g., of the xylem ele-

ments), also derives from the phenylpropanoid metabolism.

Staining with ACF (astrablue, chrysoidin, neofuchsin) revealed

that lignification of specific tissues in the inflorescence stem of

ccEe plants appeared to be diminished compared with the wild

type or the cue1 single mutants (see Supplemental Figure 6

online; Turner and Sieburth, 2003). Interestingly, the interfascic-

ular sclerenchyma cells in stems of Col-0 (see Supplemental

Figure 6A online) and cue1-6 (see Supplemental Figure 6Bonline)

showed a strong lignification, whereas the xylem elements were

only faintly stained. By contrast, there was almost no detectable

lignification of the sclerenchyma cells in stems of cue1-6/eno1-2

(+/2) (see Supplemental Figure 6C online) or cue1-1/eno1-2

(+/2) (see Supplemental Figure 6D online). In both lines, the only

lignified cells appeared to be those of the xylem elements. It is so

far unknown which factors control the degree of lignification in

the individual cell types.

Is the Growth Phenotype of the ccEe Plants Caused by

Impaired Cuticle Wax Synthesis?

In a recent report, Beaudoin et al. (2009) observed alterations in

shoots and flower development in the kcr1 mutant, which is

defective in b-ketoacyl-CoA reductase and, thus, disturbed in

fatty acid elongation. These alterations were very similar to those

observed for the ccEe plants. A complete knock out of this gene

leads to embryo lethality. Interestingly, trichomes of kcr1 ex-

hibited a distorted phenotype similar to those of the eno1 single

mutant alleles (Prabhakar et al., 2009), which is also evident for

the ccEe plants. It has been proposed that KCR1 is involved in

the synthesis of cuticle waxes and the composition of sphingo-

lipids. Toluidine blue (TB) staining of the kcr1 mutant revealed a

loss of cuticle integrity.

Wealso addressed thequestionof cuticle integrity byTBstaining

(see Supplemental Figure 7 online). Interestingly, ccEe plants

exhibited a positive staining with TB when compared with wild-

type, cue1, and eno1 plants, suggesting a lack of cuticle integrity

when the expression of ENO1 is reduced in the cue1 background.

Further insights into structural differences of the cuticle be-

tween the individual lines were obtained by scanning electron

microscopy of inflorescence stem surfaces (see Supplemental

Figure 8 online). Interestingly, the density of wax crystals was

largely diminished on stems of the ccEc plants compared with

the wild type and the cue1 and eno1 single mutants, suggesting

diminished cuticle wax deposition.

A thorough cuticle wax analysis revealed that the total wax

content was significantly increased rather than decreased in

cue1 and ccEc plants (see Supplemental Figure 9A online).

Moreover, there were some changes in the relative content of

certain wax components, such as C29 aldehyde, C27 alkane,

and C26 and C29 alcohol, which were significantly increased in

ccEe (see Supplemental Figures 9C to 9E, 9H, and 9J online) or

decreased, such as C29 ketone (see Supplemental Figure 9M

online). It is likely that such compositional changes of cuticle wax

components increase the wettability of the leaf and stem surface

and, hence, the accessibility for TB.

Chlorophyll leaching experiments, which were performed as a

further test for cuticle integrity, did not show any significant

differences between the individual lines (see Supplemental Fig-

ure 10 online). In addition, light microscopy analysis of the

adaxial leaf surface suggests a higher stomatal density in ccEe

compared with the wild-type and both single mutant plants (see

Supplemental Figure 11 online). Moreover, stomata of ccEe

appear smaller and round rather than oval shaped (see Supple-

mental Figures 11G and 11H online) compared with the wild type

and the single mutants (see Supplemental Figures 11A to 11F

online). Most relevant for TB staining, stomatal pores of ccEe

plants were widely open.

Finally, the expression of genes involved in cuticle wax bio-

synthesis, such asCER1,CER10,WAX2,BDG,KCR1, andKCR2

was not significantly affected (see Supplemental Figure 12

online). In summary, the phenotype of ccEe plants is not caused

by an impaired cuticle wax biosynthesis, hence ruling out a

shortage of lipid supply for wax biosynthesis due to the com-

bined lesions in PPT1 and ENO1.

Ectopic Overexpression of ENO1 Rescues the

cue1 Phenotype

In a previous report, we could demonstrate that ectopic over-

expression of a C4-type PPDK, driven by the CaMV 35S pro-

moter could rescue the cue1 leaf phenotype (Voll et al., 2003).

PPDK is capable of producing PEP from pyruvate and thus

replenishes PEP in plastids of those tissues where PPT1 is

missing (e.g., in cells of the vasculature of the leaf) (Knappe et al.,

2003). As ENO1 is not expressed in mature leaves (Prabhakar

et al., 2009), PEP supply relies entirely on the import by the PPT

and not on plastid glycolysis. Hence, we asked the question

2604 The Plant Cell

whether ectopic overexpression of ENO1 in the cue1-6 mutant

background is capable of rescuing the cue1 phenotype. Of

several transformed lines tested for ENO1 expression, cue1-6

ENO1 (4) and (5) showed the highest transcript abundance of

ENO1 (Figure 8B). As a control, ENO1was also overexpressed in

wild-type Col-0, yielding two lines [Col-0 ENO1 (A) and (C)] with

an increased transcript level of ENO1 (Figure 8B). As shown in

Figures 8C and 8D, ENO1 overexpressors in the cue1-6 back-

ground had indeed a wild-type-like appearance (i.e., the reticu-

late leaf phenotype was completely rescued) (Figure 8D).

However, cue1-6 transformants with a low transcript abundance

of ENO1 [e.g., cue1-6 ENO1 (1)] still showed the cue1 phenotype

(Figure 8C, 4), indicating that a threshold level of ENO1 tran-

scripts had to be exceeded to exert a rescuing effect. A similar

observation has been reported for PPDK-overexpressing lines

in the cue1-6 background (Voll et al., 2003). Moreover, over-

expression of ENO1 could also rescue the retarded growth

phenotype of cue1-6 roots (Figure 8E) but had virtually no effect

on the phenotypic appearance of roots and shoots when over-

expressed in wild-type plants (Figure 8F). These data show that

the only missing enzyme for a complete plastidic glycolytic

pathway appears to be ENO1 and not plastidic PGyM, at least in

those tissues where PPT1 is expressed in wild-type plants.

Ectopic Overexpression of ENO1 Restores Photosynthetic

Electron Transport in cue1 but Has No Effect on

Photosynthetic Performance of Wild-Type Plants

It has been shown previously that the rate of photosynthetic

electron transport (ETR) is impaired in different alleles of the cue1

mutant (Streatfield et al., 1999; Voll et al., 2003), most likely based

on decreased density of mesophyll cells characteristic of the leaf

phenotype. Hence, the rescue of the cue1 mutant phenotype by

overepressing ENO1 might restore ETR. We addressed this issue

by measuring ETR with the aid of pulse amplitude modulation

fluorometry. As shown in Figure 9, ETR in cue1-6 plants over-

expressing ENO1 recovered to wild-type levels (Figure 9B).

Moreover, there was no effect on ETR in wild-type plants

overexpressing ENO1 (Figure 9A), indicating that a complete

glycolytic pathway in chloroplasts does not interfere with ETR.

Figure 8. Ectopic Overexpression of ENO1 Rescues the cue1 Phenotype.

(A) RT-PCR with RNA extracted from leaves of wild-type (Col-0) or transformants overexpressing ENO1 in the Col-0 background.

(B) RT-PCR with RNA extracted from leaves of cue1-6 or transformants overexpressing ENO1 in the cue1-6 background.

(C) Rosette phenotypes of the wild type (1), an ENO1-overexpressing line [cue1-6 ENO1 (4)] with a high transcript abundance of ENO1 (2), the cue1-6

mutant (3), and an ENO1-overexpressing line [cue1-6 ENO1 (1)] with a low transcript abundance of ENO1 (4).

(D) Growth phenotype of flowering Col-0 wild-type (1), an ENO1-overexpressing line in the cue1-6 background [cue1-6 ENO1 (4)] with a high transcript

abundance of ENO1 (2), and cue1-6 (3) plants. The inset shows the rescue of the leaf phenotype of cue1-6 overexpressing ENO1 [line cue1-6 ENO1 (4)]

in comparison with cue1-6 (5).

(E) Rescue of the retarded root phenotype of the cue1-6mutant by overexpression of ENO1 in the cue1-6 background. Three cue1-6mutant plants are

compared with one ENO1-overexpressing line [i.e., cue1-6 ENO1 (4)].

(F) Phenotypic comparison of wild-type Col-0 with Col-0 plants overexpressing ENO1.


Overexpression of ENO1 Can Partially Rescue the

Diminished Silique and Seed Production of cue1

As ENO1 together with PPT1 is most likely involved in the

provision of PEP as precursor for fatty acid biosynthesis and for

the shikimate pathway in plastids of seeds, we determined seed

yield of the mutant plants and ENO1-overexpressing lines in the

cue1 or Col-0 background (Table 3). For the cue1-6mutant allele,

silique number, seedweight, and number of seeds per plant were

reduced significantly compared with wild-type plants. The di-

minished harvest of cue1 is most likely a consequence of

decreased maximum photosynthesis rates and retarded growth

of the shoots. Interestingly, the number of siliques and seedswas

increased in both alleles of the eno1 mutant. However, the seed

weight per plant was reduced, thus leading to an overall decline

in the specific seed weight by 20% compared with the wild type.

Two lines overexpressing ENO1 in the Col-0 background lacked

any trend of a changed yield of siliques or seeds. Line Col-0

ENO1 (C) yielded similar numbers of siliques and specific seed

weights as the wild type but exhibited lower numbers of seeds

per plant, whereas Col-0 ENO1 (A) had similar seed numbers as

the wild type but a slightly decreased specific seedweight (Table

3). The decreased number of siliques relative to thewild typewas

similar in cue1-6 plants overexpressing ENO1 [i.e., the lines

cue1-6 ENO (4) and (5)] comparedwith the cue1-6mutant plants,

despite a complete rescue of the cue1 leaf and root phenotype

(Table 3).Moreover, the number of seedswas considerably lower

in both cue1-6 ENO1 lines than in wild-type plants but was

increased compared with cue1-6, whereas the specific seed

weight in cue1-6 ENO1 was similar to wild-type plants (Table 3).

These data indicate that ectopic overexpression of ENO1 in the

wild-type background had no effect on harvest parameters,

whereas overexpression of ENO1 in the cue1 background could

partially rescue the diminished silique and seed production

characteristic for the cue1 mutant.

Seed Oil Content and Other Storage Compounds Are

Severely Diminished in ccEe Seeds

In a next step, we analyzed the storage compounds in seeds

(fatty acid content and composition as well as protein and

carbohydrate contents) of all lines used in this study (Tables 4

and 5). Total fatty acids were quantified as ameasure for seed oil

content. The total lipid content in the wild-type plants Col-0 and

Bensheim (pOCA) was 8.13 mg seed21 and 8.45 mg seed21,

respectively (Table 4). It was slightly reduced in the cue1 and

eno1 mutant alleles. This decrease was significant according to

Welch test for cue1-6 and eno1-1 but less marked in cue1-1 and

eno1-2. Of the ccEe plants, class I seeds showed a high, wild-

type-like oil content of 7 to 10 mg·seed21, while in class II seeds,

the oil content was reduced to;2.2 to 4.3 mg·seed21, and class

III seeds contained strongly reduced amounts of oil (1.9 to 2.5

mg·seed21). The strong reduction in oil content in class II and

class III seeds thus correlated with the seed size and was

accompanied by an increase in the contents of saturated fatty

Figure 9. Light Saturation Curves of Photosynthetic ETRs Determined

by Imaging Pulse Amplitude Modulation Fluorometry.

(A) Comparison of light saturation curves between Col-0 (open circles),

eno1-2 (triangles), Col-0 ENO1 (A) (closed circles), and Col-0 ENO1 (C)

(squares).

(B) Comparison of light saturation curves between Col-0 (cirlces), cue1-6

(squares), and cue1-6 ENO1 (4) (triangles).

The data represent the mean 6 SE of n = 9 measurements on individual

plants per line.

Table 3. Harvest Parameters of Wild-Type Arabidopsis (Col-0), cue1-6, and ENO1-Overexpressing Lines in the Col-0 or cue1-6 Background

Plant Line Siliques per Plant

Seed Weight per

Plant (mg)

Specific Seed Weight

(mg·seed�1) Number of Seeds per Plant

Col-0 208.5 6 10.2 (100) 104.2 6 5.9 (100) 14.4 6 0.7 (100) 7239

cue 1-6 188.7 6 6.7 (91) 49.0 6 2.4 (47)a 12.4 6 0.5 (86) 3941

Col-0 ENO1 (A) 201.7 6 7.6 (97) 79.5 6 5.9 (76)b 14.5 6 1.1 (101) 5466

Col-0 ENO1 (C) 199.4 6 5.4 (96) 89.8 6 7.7 (79) 12.4 6 0.6 (86)d 7266

cue 1-6 ENO1 (4) 180.5 6 9.0 (87)d 79.1 6 4.1 (76)b,a 13.2 6 0.2 (92) 5989

cue 1-6 ENO1 (5) 186.2 6 12.4 (88) 82.1 6 7.0 (86)c,a 14.0 6 0.6 (97)d 5875

Plants were grown in a temperature-controlled greenhouse during Febuary and March. The data are expressed as mean 6 SE. The specific seed

weight was estimated from 100 to 200 seeds counted. The data represent the mean value 6 SE of 15 individual plants per line. Statistical significance

of differences between the parameters was assessed by the Welch test with probability values of P < 0.001 (a), P < 0.01 (b), P < 0.02 (c), and P < 0.05

(d). The bold letters in italics refer to cue1-6 as a control.

2606 The Plant Cell

acids (i.e., 16:0, 18:0, and 22:0), while the amounts of unsatu-

rated fatty acids (i.e., 18:3 and 20:1) decreased (Table 4). The

strong decrease in 20:1 content is indicative of a reduction in the

amount of storage lipids versus membrane lipids in the seeds

because 20:1 is restricted to the triacylglycerol pool. These

changes are also reflected in a strong decrease in the desatu-

ration index (ID), which indicates the number of double bonds in

all unsaturated fatty acid classes divided by the number of all

saturated fatty acid classes. Furthermore, the increase in 16:0

and the decrease in 18:3 affected the ratio of C16 to C18 fatty

acids (for a complete comparison of fatty acid composition, see

Supplemental Figure 13 online). Overexpression of ENO1 in the

Col-0 background had no severe effect on lipid content and

composition. The average lipid content was slightly less in Col-0

ENO1. This was supported by an increase in the C16/C18 ratio

and a decrease in ID suggesting a diminished rather than an

enhanced production of storage lipids.

MatureArabidopsis seeds store similar amounts of protein and

lipids when as a percentage of the dry weight (Chen et al., 2009).

As shown in Table 5, total protein content was similar in the single

cue1 and eno1mutants compared with the respective wild-type

or control plants, leading to a decline in the lipid/protein ratio by

10 to 20% in the cue1 alleles and up to 30% in the eno1 alleles

(Table 5). These data indicate that oil content rather than protein

content responds more strongly when PPT1 and ENO1 are

impaired. Similar to the seed oil content, protein content was

severely diminished in class III seeds of the segregating ccEe

plants, whereas class II seeds showed an intermediate decline in

protein content.

Seeds of Arabidopsis contain sucrose as major carbohydrate.

In mature seeds, starch was below the detection limit of the

coupled enzymatic assay applied. Total carbohydrate contents

were not appreciably affected in the single mutants compared

with the respective wild-type plants (Table 5). Similar to seed oil

and protein, sucrose content was severely diminished in class III

and intermediately reduced in class II seeds of the ccEe plants.

Interestingly the levels of both hexoses (glucose and fructose)

were appreciably increased in the strong cue1 alleles aswell as in

both eno1 mutant alleles relative to the respective wild-type

plants. Likewise, contents of bothhexoseswereenhanced in class

III seeds of the ccEe plants, whereas class II seeds contained

diminished hexose contents relative to the wild-type-like class I

seeds. It is conceivable that a block in oil production leads to a

diminished sucrose consumption by glycolysis (Lonien and

Schwender, 2009) and results in enhanced hydrolytic cleavage

of sucrose by invertase activities. Again, overexpression of ENO1

in the Col-0 or cue1-6 backgrounds had no strong effect on seed

protein and carbohydrate contents. Increased carbohydrate con-

tents have been observed in seeds of thewri1mutant, defective in

seed carbohydrate use (Focks and Benning, 1998; Lonien and

Schwender, 2009).

DISCUSSION

In this report, we analyzed the central role of PEP in plant

metabolism with the aid of Arabidopsismutants impaired in PEP

provision to the plastids. For this purpose, we crossed three

Table 4. Lipid Contents and Fatty Acid Composition in Seeds of Wild-Type Arabidopsis (Col-0 and pOCA), eno1 and cue1 Alleles, and

ENO1-Overexpressing Lines in the Col-0 or cue1-6 Background and Heterozygous eno1Mutants in the Homozygous cue1 Background (ccEe Plants)

Plant Line Total Lipid (mg·Seed�1) C16/C18 Ratio C20:1 (Mol %) ID n

pOCA 8.45 6 0.29 0.123 13.60 6 0.28 11.15 5

cue1-1 6.63 6 0.73 0.112 13.08 6 0.47 12.91 5

cue1-3 6.77 6 0.44c 0.109 13.96 6 0.47 13.35 5

Col-0 8.13 6 0.27 0.104 13.56 6 0.23 10.71 10

cue1-6 5.70 6 0.35a 0.112 12.85 6 0.32 12.41 5

eno1-1 5.53 6 0.29a 0.142 12.98 6 0.27 10.02 10

eno1-2 7.77 6 0.49 0.132 13.26 6 0.18 10.28 10

Col-0 ENO1 7.17 6 0.55 0.154 12.63 6 0.27d 9.37 10

cue1-6 ENO1 7.21 6 0.30d,b 0.137 13.49 6 0.29 9.88 10

cue1-1/eno1-1(+/�) (class III) 2.51 6 0.41b 0.208 8.92 6 0.72b 5.56 5

cue1-1/eno1-1(+/�) (class II) 3.98 6 0.44d 0.141 10.83 6 0.66 8.54 5

cue1-1/eno1-1(+/�) (class I) 10.40 6 0.27b 0.099 14.03 6 0.29 13.02 5

cue1-1/eno1-2(+/�) (class III) 2.10 6 0.20b 0.278 4.24 6 0.52a 3.45 5

cue1-1/eno1-2(+/�) (class II) 2.25 6 0.32b 0.284 5.49 6 0.55a 3.26 5

cue1-1/eno1-2(+/�) (class I) 6.50 6 0.13 0.121 11.67 6 0.25 8.75 5

cue1-3/eno1-2(+/�) (class III) 2.18 6 0.18a 0.249 7.51 6 0.61a 3.98 5

cue1-3/eno1-2(+/�) (class II) 4.26 6 0.37b 0.162 10.35 6 0.74c 6.52 5

cue1-3/eno1-2(+/�) (class I) 7.00 6 0.23 0.119 12.53 6 0.20d 9.76 5

cue1-6/eno1-2(+/�) (class III) 1.91 6 0.19a 0.323 6.01 6 0.95b 2.80 5

cue1-6/eno1-2(+/�) (class II) 2.62 6 0.15a 0.287 8.53 6 0.73c 3.90 5

cue1-6/eno1-2(+/�) (class I) 7.07 6 0.26d 0.132 11.62 6 0.05c 8.76 5

Lipid contents and fatty acid composition determined on individual seeds (n = 5 to 10) and C16/C18 ratios as well as the desaturation index (ID) were

calculated from the mol % of individual fatty acids shown in Supplemental Figure 13 online. The data represent the mean value 6 SE. Statistical

significance of differences between the parameters was assessed by the Welch test with probability values of P < 0.001 (a), P < 0.01 (b), P 0.02 (c), and

P < 0.05 (d). Bold letters in italics refer to cue1-6 as a control.


different alleles of the cue1 mutant (defective in PPT1; Li et al.,

1995; Streatfield et al., 1999; Knappe et al., 2003; Voll et al., 2003)

with two alleles of the eno1 mutant (Prabhakar et al., 2009).

Interestingly, plants lacking both the PPT1 and ENO1 genes

could not be obtained as double homozygous lines and even

heterozygous eno1 mutants in the homozygous cue1 back-

ground exhibited a severe dwarfish phenotype combined with

aberrant flower, silique, and seed development. This result was

surprising asArabidopsis contains a second functionalPPT gene

(i.e., PPT2), which is expressed, apart from the roots, in most

vegetative and generative tissues (see Supplemental Table 3 and

Supplemental Figure 14 online). Moreover, Arabidopsis contains

aPPDK gene,which could providePEP frompyruvate in plastids.

However, neither PPT2 nor PPDK is capable of compensating for

the deficiencies in PPT1 and ENO1; thus, normal development of

Arabidopsis plants depends on the presence of these two genes.

Furthermore, it is surprising that a heterozygous defect in the

ENO1 gene in the cue1 background has such a strong impact on

whole-plant development, in particular as ENO1 is not ex-

pressed, for instance, in mature leaves. Hence, it is likely that

fully functional PPT1 and ENO1 activities are required in early

plant development (i.e., in the apical and leaf meristems, where

both genes are highly expressed) (Prabhakar et al., 2009). A

detailed analysis of the spatial and temporal expression pattern

of genes involved in PEP and pyruvate metabolism in plastids is

contained in Supplemental Table 3 and Supplemental Figure 14

online based on publicly available microarray data and the aid of

the efp browser of the University of Toronto (http://bar.utoronto.

ca/efp/cgi-bin/efpWeb.cgi; Winter et al., 2007).

In addition, we could rescue the cue1 phenotype by constitu-

tive overexpression of ENO1, indicating that (1) a full comple-

ment of plastidial glycolytic enzymes can provide a sufficient flux

into PEP to compensate for a deficiency in its import by PPT1 and

(2) that ENO, but not PGyM, is the only missing enzyme for a

complete glycolysis in chloroplasts and root plastids. However,

overexpression of ENO1 could not improve seed yield and seed

oil contents in wild-type plants.

The Segregation of cue1 and eno1 Revealed Constraints in

Gametophyte and Sporophyte Development

The offspring of crosses between cue1 and eno1, using three

alleles of the former and two alleles of the latter, did not yield

double homozygotes, indicating that lethality of the ccee plants is

independent of the ecotypes (i.e., Col-0 or Bensheim) or second-

ary mutations. Moreover, even the percentage of heterozygous

eno1mutants in the homozygous cue1 background (ccEe plants)

was far below expectations (Table 1). Likewise, the percentage of

heterozygous cue1 mutants in the homozygous eno1 (Ccee

plants) background was less than half the expected number (see

Supplemental Table 1B online). Strikingly, ccEe plants exhibited a

pronounced phenotype in the sporophyte, including growth retar-

dation, deformed flowers, and shortened siliques (Figure 2),

whereas the sporophyte of Ccee plants was not noticeably

compromised during vegetative development. As ENO1 expres-

sion was decreased in those vegetative tissues where ENO1

transcripts were highly abundant in wild-type plants (i.e., in roots

and the shoot apex), the constraints in vegetative development in

ccEe plants were most likely due to a diminished function of the

ENO1protein. Such sporophytic constraintswere also reflected in

the male and female TE (see Supplemental Table 1C online)

calculated from the genotype distribution in the offsprings of

reciprocal crosses between cue1-1 (male)3 eno1-2 (female) and

eno1-1 (male) 3 cue1-1 (female). For the cue1-1 mutation, the

male and female TEs of;30% were very similar, whereas for the

eno1-2 mutation, the female TE was decreased to ;15% com-

paredwith themale TE,which againwas close to 30%.Hence, the

additional decline in the female TE for the eno1-2 mutation

suggests sporophytic rather than gametophytic constraints.

Restrictions in the Shikimate Pathway and in Fatty Acid

Biosynthesis Are the Main Reasons for Impaired

Gametophyte Development

The underlying reasons for developmental constraints of female

(Figure 3) and male gametophytes (Figure 4) in ccEe plants are

Table 5. Protein and Carbohydrate Contents in Seeds of Wild-Type Arabidopsis (Col-0 and pOCA), eno1 and cue1 Alleles, and ENO1-

Overexpressing Lines in the Col-0 or cue1-6 Background and Heterozygous eno1 Mutants in the Homozygous cue1 Background (ccEe Plants)

Plant Line

Total Protein

(mg·Seed�1) Lipid/Protein Ratio Sucrose Glucose (ng·Seed�1) Fructose

Seed Weight

(mg·Seed�1)

pOCA 10.28 6 0.23 0.82 308 6 31 5.10 6 0.66 4.23 6 0.44 33.33 6 3.43

cue1-1 10.60 6 0.96 0.63 277 6 10 7.61 6 1.34 7.02 6 1.01d 15.72 6 1.67d

cue1-3 10.98 6 0.22 0.62 309 6 4 4.99 6 0.94 3.92 6 1.72 26.67 6 1.91

Col-0 10.56 6 0.31 0.77 249 6 13 2.34 6 0.60 2.39 6 0.51 30.00 6 1.17

cue1-6 7.74 6 0.18b 0.74 374 6 24c 9.24 6 4.14 7.58 6 2.20 24.76 6 1.91

eno1-1 10.95 6 0.12 0.51 387 6 24b 9.42 6 1.98 7.02 6 2.30 36.19 6 0.95c

eno1-2 10.42 6 0.29 0.75 317 6 25 12.76 6 1.07b 8.23 6 0.16b 22.86 6 2.86

Col-0 ENO1 10.74 6 0.44 0.67 341 6 33d 4.70 6 0.60d 3.91 6 0.87 28.57 6 3.76

cue1-6 ENO1 9.86 6 1.07 0.73 307 6 14c 5.65 6 0.91d 4.21 6 0.60d 28.10 6 3.79

cue1-1/eno1-2(+/�) (class III) 1.50 6 0.35a 1.67 22 6 1b 6.88 6 1.79 10.51 6 1.70d 7.62 6 0.95a

cue1-1/eno1-2(+/�) (class II) 4.83 6 0.55b 0.82 177 6 8d 2.45 6 0.64d 3.17 6 0.74 13.33 6 1.91b

cue1-1/eno1-2(+/�) (class I) 12.14 6 0.38c 0.86 293 6 10 6.59 6 1.10 5.21 6 1.21 27.62 6 5.04

The contents of protein and carbohydrates (sucrose, glucose, and fructose) were referred to individual seeds of mutant and wild-type plants. The

individual seed weight was determined in three to six batches of 35 seeds per line. The data represent the mean value 6 SE. Statistical significance of

differences between the parameters were assessed by the Welch test with probability values of P < 0.001 (a), P < 0.01 (b), P < 0.02 (c), and P < 0.05 (d).

2608 The Plant Cell

based most likely on a restriction of metabolism starting from

plastidial PEP.

While PEP imported into nongreen plastids by the PPT has to

be generated by cytosolic glycolysis and/or gluconeogenesis, its

formation inside the plastid ought to commence from imported

Glc6P or 3-PGA and the subsequent conversion to PEP by

plastid glycolysis involving PGyM and ENO1 (Figure 1A). It has

been been shown previously that Glc6P can enter the plastid via

one of the two Glc6P/phosphate translocators (GPTs) of Arabi-

dopsis (Kammerer et al., 1998). Arabidopsismutants defective in

GPT1 could not be obtained as homozygous lines (Niewiadomski

et al., 2005). In the heterozygous gpt1 mutant, both female and

male gametophytes show high rates of lethality comparable to

those of the ccEe plants. For developing pollen of the heterozy-

gous gpt1mutant, it could be shown that, besides the absence of

starch, oil production seemed to be also diminished in pheno-

typically modified pollen. As Glc6P is required for the OPPP

(Kruger and von Schaewen, 2003), by which reducing equiva-

lents in form of NADPH are provided for anabolic reaction

sequences, such as fatty acid biosynthesis, the absence of

GPT1 in the haploid gametophytes was proposed to lead to a

deficiency in membrane formation and eventually death

(Niewiadomski et al., 2005). Hence, NADPH supply by the

OPPP for fatty acid biosynthesis appears to be crucial for early

gametophyte development.

Here, we could show that, in turn, a restriction in PEP supply to

plastids leads to a high lethality rate of gametophytes in ccEe

plants. It is tempting to speculate that, at least in the female

gametophytes PEP, after conversion to pyruvate, serves as the

major substrate for fatty acid biosynthesis. Hence, diminished

fatty acid provision may lead to a halt in ovule development in

ccEe plants (Figure 3) similar to the heterozygous gpt1 mutant

(Niewiadomski et al., 2005).

However, diminished fatty acid supply cannot explain the high

lethality rate ofmale gametophytes in ccEeplants. Ultrastructural

analysis revealed that pollen of the ccEe plants contained at least

similar numbers of lipid bodies and vacuoles compared with

wild-type pollen (Figure 5), indicating that fatty acid biosynthesis

is not likely to be restricted during male gametophyte develop-

ment in ccEeplants. It is hence conceivable that lethality of pollen

in the ccEe plants is rather due to a restriction in the shikimate

pathway. This notion is supported by autofluorescence imaging

of phenolic compounds in pollen sacs (see Supplemental Figure

2 online) and individual pollen of ccEe plants compared with the

wild type (see Supplemental Figure 3 online). In both cases,

phenolic compounds were severely diminished. Moreover, flow-

ers of ccEe plants contained significantly decreased levels of

flavonoids (see Supplemental Table 2 online), again indicating a

restriction of the shikimate pathway in floral organs.

The exine structure, which is strongly defective in pollen of

ccEe plants, consistsmainly of sporopollonin, which is formedby

the diploid cells of the pollen sac secretory tapetum (Ariizumi

et al., 2004). Sporopollonin is an extremely rigid substance

containing both long-chain fatty acids and phenolic compounds

derived from the phenylpropanoid metabolism (Guilford et al.,

1988; Wiermann et al., 2001). Hence, the impaired exine forma-

tion observed in the majority of the pollen (80%) in ccEe plants is

most likely due to a diminished gene dosage of ENO1 in the

absence of PPT1 in the tapetum cells rather than an absence of

both proteins in the microspores. The heterozygous knockout of

ENO1 in the cue1 background might thus hamper sporopollonin

production by the tapetum cells due to diminished PEP provision

for the shikimate pathway in the plastids therein.

However, an exine phenotype similar to that observed for

pollen from ccEe plants has recently been reported for an

Arabidopsis mutant defective in CYP704, a cytochrome P450–

dependent long-chain fatty acid v-hydroxylase involved in

sporopollonin biosynthesis (Dobritsa et al., 2009), indicating

that in the case of the cyp704 mutant, an impaired provision of

long-chain fatty acids by the pollen sac tapetum compromises

exine formation.

Seed Development in ccEe Plants Is Impaired Due to a

Restriction in Fatty Acid Biosynthesis

Unlike plastids of nongreen tissues, plastids of developing oil

seeds aremixotrophic and obtain substantial parts of their energy,

reducing power and 3-PGA by photosynthesis (Ruuska et al.,

2004; Li et al., 2006). In knockdownmutants or antisense plants of

plastid-localized PK, seed development was severely compro-

mised (Andre et al., 2007; Baud et al., 2007b), indicating that

pyruvate as precursor for fatty acid biosynthesis is provided from

PEP inside the plastids rather than by import from the cytosol.

Moreover, the wri1 mutant, defective in a transcription factor

(Cernac andBenning, 2004) that regulates the expressionof genes

involved in carbohydrate metabolism (e.g., glycolysis), showed a

similar phenotype (Focks and Benning, 1998; Baud and Graham,

2006; Baud et al., 2007a). This aspect of embryo development has

recently been further analyzed by elegant 13C flux studies (Lonien

and Schwender, 2009) with mutant plants compromised in PKp

(Baudet al., 2007b) orwithwri1 (FocksandBenning, 1998;Ruuska

et al., 2002; Baud et al., 2007a). Both pkp1 pkp2 double mutants

and wri1 exhibit a severe decrease in seed oil contents. Hence, in

Col-0 wild-type plants, the majority (89%) of plastidic pyruvate

used for fatty acid biosynthesis in seeds was derived from PKp,

and the residualmight be shared by pyruvate import and oxidative

decarboxylation by plastidic ME4. In our approach, seed oil

content was severely diminished in class II and III seeds of the

segregating ccEe plants, which were heterozygously mutated in

the ENO1 gene in the background of the homozygous cue1

mutants (cf. Table 4), thus supporting the significance of PEP

supply to plastids for seed oil production.

The Phenotype of ccEe Plants Is Not Connected to a

Restriction in Cuticle Wax Biosynthesis

At a first glance, the sporophytic and gametophytic phenotype of

ccEe plants resembled in many aspects mutant plants impaired

in fatty acid elongation, such as kcr1, defective in a b-ketoacyl-

CoA reductase (Beaudoin et al., 2009). Homozygous kcr1 mu-

tants were embryo lethal, and siliques of heterozygous kcr1

mutants contained 25% aborted seeds. The remaining seeds

appeared white and transparent. To analyze the function of

KCR1 during vegetative development, Beaudoin et al. (2009)

created RNA interference (RNAi) plants, which exhibited a variety

of phenotypes, including deformed flowers, stunted growth of


rosette leaves, as well as shortened and crooked siliques. These

are very similar to the phenotypes of ccEe plants. Interestingly

KCR1 RNAi plants also showed deformed trichomes similar to

those of the eno1 single mutants (Prabhakar et al., 2009) and

ccEe plants. Staining with TB and additional experimental

approaches revealed that the phenotype of the KCR1 RNAi

plants was most likely due to disturbed cuticle wax biosynthesis

(Beaudoin et al., 2009).

Here, we observed a similar leakiness of the cuticle in leaves

and stems of ccEe plants (see Supplemental Figure 7 online),

suggesting impaired cuticle wax biosynthesis. In parallel to the

enhanced TB staining, we found decreased density in epicutic-

ular wax of stems (see Supplemental Figure 8 online). Surpris-

ingly, wax amounts were not altered or even increased in ccEe

plants (see Supplemental Figure 9 online). However, comparable

observations have been made with other cuticle mutants in the

past. Increased chlorophyll leaching from leaves and enhanced

TB staining of leaves in mutants compared with the wild type

were accompanied by significant increases in wax amounts

(Aharoni et al., 2004; Schnurr et al., 2004; Kurdyukov et al., 2006)

and not by decreases as could intuitively be expected. This leads

to the conclusion that it must be ultrastructural changes in wax

arrangement or in wax deposition to the cutin polymer probably

combined with an increased stomatal aperture (see Supplemen-

tal Figure 11 online) leading to the observed effects of enhanced

cuticular permeability and not necessarily decreases in wax

amounts. These data also suggest that a restriction in fatty acid

biosynthesis in the apical meristem, which would lead inevitably

to a restriction in cuticle wax biosynthesis starting from stored

oleosomes (Panikashvili and Aharoni, 2008), is not the main

reason for the developmental phenotype of ccEe plants.

The Retarded Growth Phenotype of ccEe Plants Might

Originate from an Increased Jasmonate Content and

Disturbed Amino Acid Metabolism

To further elucidate the underlying reasons for the retarded

growth phenotype of ccEe plants, the spectrum of proteinogenic

free amino acids and phytohormones was determined in rosette

leaves and flowers of wild-type, single mutant, and ccEe plants.

Interestingly, leaves of both cue1 and ccEe plants contained

elevated levels of total free amino acids, due to increased

amounts of the major amino acids. By contrast, contents of

minor amino acids, which in principle derive from plastidial PEP,

such as aromatic and branched-chain amino acids, were se-

verely diminished on a relative but not on an absolute scale.

Similar changes in the amino acid spectrum have recently been

reported for the Phe insensitive growth (pig) mutant ofArabidopsis

(Voll et al., 2004).However, the underlyingmolecular reason for the

disturbed amino acid composition in the pigmutant has not been

further resolved. Strinkingly Ala, His, and Arg were dramatically

increased in leaves of ccEe plants both on a relative and absolute

basis. Arg has been proposed to act as a precursor for the

synthesis of NO in plants (Guo et al., 2003), which exerts multiple

effects on plant growth and development (del Rio et al., 2004).

Unlike in rosette leaves, in flowers, total free amino acid

remained unchanged in the single mutants and the ccEe plants

compared with the wild type. Of the aromatic amino acids, only

Trp was significantly decreased in cue1-6/eno1-2(+/2). Since

Trp can serve as a precursor for auxin biosynthesis (Bartel, 1997),

the stunted growth of the shoot and constraints in early flower

development could hence also be linked to modified auxin

availability (Pagnussat et al., 2009; Vanneste and Friml, 2009).

However, the auxin IAA decreased in flowers of cue1-6 and ccEe

plants, whereas its content remained nearly unchanged in leaves

of the same lines (Figure 7). Hence, diminished auxin contents

can be ruled out as an underlying reason for the developmental

constraints in ccEe plants. Interestingly, the eno1-2 single mu-

tant exhibited a significant increase in IAA, ABA, and SA con-

tents. However, increased levels of these compounds did not

result in any obvious growth phenotype of the eno1mutant. ABA

levels were only slightly but significantly enhanced in flowers of

ccEe plants compared with the wild type and the single mutants.

However, it appears unlikely that the moderate increase in ABA

levels is the major reason for constraints in flower development.

In rosette leaves, but not in flowers, contents of JA and its

precursor oPDA exhibited a significant fourfold increase in ccEe

compared to wild-type plants. It has been demonstrated that an

increase in jasmonate levels causes growth retardation (e.g., in

the cev1mutant defective in the cellulose synthase CeSA3) (Ellis

et al., 2002) or in the fatty acid oxygenation upregulated8mutant

(Rodrıguez et al., 2010). Wound-induced increased JA levels

inhibit mitosis and thus result in stunted growth or a bonsai

phenotype of the shoot (Zhang and Turner, 2008). It is hence

likely that increased JA levels are the major reason for growth

retardation of ccEe plants. However, as JA derives from fatty

acid metabolism, an increase in its content, as observed here,

cannot be directly linked to the lesion in PPT1 and a decline in

ENO1.

Cytokinin levels were increased in rosette leaves of both cue1

and ccEe plants. Pyruvate delivered from plastidic PEP by the

action of PKp can enter the biosynthesis of isoprenoids via

theMEP pathway. TheMEP pathway has been shown to provide

the prenyl group of the cytokinins trans-zeatin and isopentenyl

adenine via plastid-localized isopentenyltransferases (Kasahara

et al., 2004). However, an impairment of the MEP pathway in

ccEe plants would most likely lead to diminished rather than

increased cytokinin levels.

Considering the expression profile of ENO1 and PPT1 (see

Supplemental Table 3 and Supplemental Figure 14 online), it is

hard to conceive as to why a heterozygous lesion in ENO1 in the

background of the cue1 mutant has such a dramatic effect on

shoot development in Arabidopsis. Unlike PPT1, ENO1 is not

expressed in mature leaves (see Prabhakar et al., 2009). How-

ever, both genes share expression in the apical shoot meristem

and in the meristem of young leaves (Prabhakar et al., 2009).

Hence, we conclude that a disturbance of the developmental

program rather than in metabolism is the major cause for the

retarded growth phenotype in ccEe plants. In leaves, PPT1

expression is found in the vasculature, including the bundle

sheath. In particular, PPT1 is highly expressed in xylem paren-

chyma cells (Knappe et al., 2003), which led to the assumption

that certain metabolic signals deriving from the phenylpropanoid

pathway are generated in these cell types and may control

mesophyll development (Voll et al., 2003). The link of the defi-

ciency of PPT1 in vegetative tissues and the occurrence of the

2610 The Plant Cell

reticulate leaf phenotype of the cue1 mutant remains obscure

and is currently the subject of intense investigations.

Constitutive ENO1Overexpression Rescues the

cue1 Phenotype

It has previously been shown that constitutive overexpression of

the C4-type PPDK of Flaveria trinervia targeted to the plastids

rescued the reticulate leaf phenotype of the cue1 mutant (Voll

et al., 2003). Thus, a limitation of PEP import into the stroma via

PPT1 can be compensated by PEP generation from pyruvate

within the plastids. As ENO1 is not expressed in mature leaves

(see Supplemental Table 3 and Supplemental Figure 14 online;

Prabhakar et al., 2009), PEP provision by plastid-localized gly-

colysis can be excluded. Here, we could demonstrate that

constitutive overexpression of ENO1 could rescue both the leaf

and root phenotypes of cue1. Thus, our data demonstrate (1) that

ENO1 is the only missing enzyme for a complete glycolytic

pathway within plastids, at least in those cells where PPT1 is

expressed, and (2) that sufficient flux through plastid glycolysis

exists to compensate the restriction in PEP import into the

stroma by PPT1. It is likely that in chloroplasts and in plastids

from heterotrophic tissues, which lack ENO1 expression, gly-

colysis ceases at the step of 3-PGA to 2-PGAconversion (i.e., the

reaction catalyzed by plastid-localized PGyM).

According to the ARAMEMNON database (http://aramemnon.

botanik.uni-koeln.de/; Schwacke et al., 2004), the genome of

Arabidopsis contains 24 putative PGyM genes. Of the 24 PGyM

genes, there are six genes encoding proteins with a strongly

predicted N-terminal transit peptide for plastid targeting

(At1g22170, At1g58280, At1g78050, At3g52155, At5g22620,

and At5g62840). Whereas At1g22170 and At1g78050 are not

expressed in photosynthetic active tissues, the transcripts of of

the other four are also abundant in leaves, suggesting that 2-PGA

can be formed also in chloroplasts. It has been demonstrated

that both PPTs of Arabidopsis are capable of a 2-PGA/PEP

counterexchange (Knappe et al., 2003). It is hence conceivable

that the PPT (PPT1 and/or PPT2) controls to certain extent the

flux of photoassimilates into secondary metabolism (i.e., the

shikimate pathway) by sensing the metabolic status within

the mesophyll. Under conditions that favor the accumulation of

3-PGA and 2-PGA (catalyzed by PGyM) in the chloroplast stroma

(e.g., as a consequence of sink limitation in the cytosol), 2-PGA/

PEP counterexchange by the PPT would increase and hence

open theway in the direction of the production of aromatic amino

acids and follow-up products. As has recently been shown for

tobacco (Nicotiana tabacum) plants with an antisense inhibition

of a cytosolic ENO, a limitation in cytosolic PEP production

results in a reticulate leaf phenotype similar to that of cue1,

suggesting that in wild-type plants, cytosolic PEP supply is

sufficient (Voll et al., 2009).

Constitutive overexpression of ENO1 in wild-type plants had

neither an obvious effect on the final size of the transformants

and leaf morphology nor on the photosynthetic capacity. How-

ever, amore detailed analysis is required to address the question

as to whether a complete glycolytic pathway within the chloro-

plast stroma fine-tunes primary and secondary metabolism.

In chloroplasts, the glycolytic sequence involving PGyM and

ENO, for instance, would withdraw 3-PGA fixed by ribulose-

1,5-bisphosphate carboxylase/oxygenase in the light and could

hence result in a depletion of Calvin cycle intermediates com-

bined with a decline in the production of photoassimilates.

Concluding Remarks

Our data clearly show that both PPT1 and ENO1 are indispens-

able for survival of Arabidopsis plants and demonstrate that

plastidial PEP plays a crucial role for normal plant development.

In the future, it will be challenging to gain more information on the

fine-tuning of plastid-to-cytosol communication involving plas-

tidial transport proteins, such as the pyruvate transporter and

PPT2. For both transporters, there is lack of information on their

functional involvement in plant development and metabolism

because (1) the pyruvate transporter has not yet been identified

and (2) there are no PPT2 knockout mutants available to date.

METHODS

Plant Material and Growth Conditions

Wild-type and mutant Arabidopsis thaliana plants, ecotype Col-0 or the

transgenic control plant pOCA106, ecotype Bensheim (Li et al., 1995),

were used in all experiments. The cue1 alleles (cue1-1, cue1-3, and cue1-6)

were isolated in a genetic screen based on the aberrant expression of

CAB3 (Li et al., 1995; Streatfield et al., 1999). The eno1 mutant alleles

(eno1-1, N521328; and eno1-2, N421734) were provided by the Notting-

ham Arabidopsis Stock Centre (http://Arabidopsis.info/). Arabidopsis

plants were germinated and grown on soil for 4 to 5 weeks in a

temperature-controlled greenhouse at a 17-h-light/7-h-dark cycle. The

average photon flux density on plant level was 70 to 100 mmol·m2·s1. For

growth of plants in sterile culture, seeds were surface sterilized and

germinated on agar supplemented with half-strength MS medium con-

taining 2% sucrose (Duchefa). Plantlets were grown in a Percival growth

cabinet at day/night temperatures of 218C/188Cand a 17-h-light/7-h-dark

cycle at an average photon flux density of 50 to 80 mmol·m2·s1.

Generation of cue1 eno1 Double Mutants and Segregation Analysis

Immature flowers of homozygous single mutants were emasculated and

manually cross-pollinated. The crosses of cue1-1 3 eno1-1, cue1-1 3

eno1-2, cue1-3 3 eno1-2, and cue1-6 3 eno1-2 were performed using

the eno1-1 or eno1-2 plants as the female parent. In the reciprocal cross

of eno1-23 cue1-1, the cue1-1 plant was used as the female parent. The F1

plants heterozygous for both alleles were selfed, and the heterozygous

doublemutantswere identifiedbygenotyping in theF2or higher populations.

Genotyping of eno1Mutants by PCR

Wild-type andmutant alleles of ENO1were identified by PCR on genomic

DNA extracted according to Edwards et al. (1991). The insertion site of the

T-DNA from the GABI-Kat collection (Rosso et al., 2003) was identified

using the T-DNA–specific primer GABI LB pAC161 08409 (59-ATATT-

GACCATCATACTCATTGC-39) in combination with the appropriate

gene-specific primers, for example, the primer pairs ENO1-1 (sense)

59-CAGGATGATTGGAGCTCATGG-39 and ENO1-1 (antisense) 59-CGA-

CATATCTCTGAGCATCTG-39 for the eno1-1 mutant allele and ENO1-2

(sense) 59-ATCATGTCTGAGATTCCGTCG-39 and ENO1-2 (antisense)

59-CTGACCTTCACTTCCCATCTG-39 for the eno1-2 mutant allele. For

genotyping of the seed classes I, II, and III of the ccEc plants, 50 seeds of


each class was selected and DNA extracted by the Extract-N-Amp-Seed

kit (Sigma-Aldrich).

Genotyping of cue1Mutants in the T3 Generation

The mutant alleles cue1-6 and cue1-1 were obtained from ethyl meth-

anesulfonate–mutagenized seeds or after g-ray treatment (deletion of the

PPT1 locus), respectively. The hetero- or homozygous T-DNA insertion in

the ENO1 gene was analyzed by PCR on genomic DNA, whereas hetero-

zygous mutations in the PPT1 gene were tested in the progeny of the F2

plants after self-fertilization. Those plants that were heterozygous for cue1

in the F2 generation again segregated in the F3 generation and exhibited

homozygous cue1 plants with its characteristic phenotype. Moreover, as

mutant plants homozygous for the mutation in both genes could not be

isolated, the occurrence of a heterozygous mutation in the PPT1 gene in

the homozygous eno1mutant backgroundwas analyzed bypollen viability

tests in the F3 generation after self-fertilization of the F2 plants. Only those

plants showing a pollen abortion of >10% in the F3 generation were

considered to have been heterozygous for cue1 in the F2 generation.

TE

The TE of reciprocal crosses of cue1-1 and eno1-2 was calculated

according to Blanvillain et al. (2008) and is defined as the number of

mutated alleles divided by the number of total alleles times 100.

Expression Analysis

For RT-PCR analysis, total RNA was isolated from the individual lines

using the RNA plant mini kit (Qiagen) according to the manufacturer’s

instruction. Oligo(dT)-primed cDNA from 2 mg of total RNA (DNase

treated) was synthesized using the Bioscript reverse transcriptase sys-

tem (Bioline). ENO1 was amplified with the primer pair ENO1 (sense)

59-CACCATGGCTTTGACTACAAAACC-39 and ENO1 (antisense) 59-TCA-

TGGTGATCGGAAAGCTTCACC-39. Actin2 served as a control with the

primer pair Actin (sense) 59-TAACTCTCCCGCTATGTATGT-39 and Actin

(antisense) R 59-CCACTGAGCACAATGTTACCGTAC-39. The amplifica-

tions was performed with the following settings, 948C for 5 min followed

by 35 cycles at 94, 55, and 728C, 30 s each, and a final extension at 728C

for 7 min.

For qRT-PCR analyses, total RNA was isolated using the RNAeasy

Plant mini kit (Qiagen) according to the manufacturer’s protocol, and

cDNA was synthesized from 2 mg of total RNA as described above. The

qRT-PCR reactions were conducted in the presence of the Power SYBR

Green master mix (ABI Prism) according to the manufacturer’s instruc-

tions in a 7300 Real-Time PCR system (Applied Biosystems) equipped

with Sequence Detection Software v1.4. Expression levels of candidate

genes were normalized to Actin2 expression levels as a control. The qRT-

PCR experiments were done on three independent biological samples

and three technical replicates. The primer sequences of genes used in

qRT-PCR experiments are listed in Supplemental Table 4 online, includ-

ing those involved in cuticle wax biosynthesis, such as CER1 (Aarts et al.,

1995), WAX2 (Chen et al., 2003), CER10/ECR (Zheng et al., 2005), BDG

(Kurdyukov et al., 2006), KCR1, and KCR2 (Beaudoin et al., 2009).

Overexpression of ENO1

Plants constitutively overexpress ENO1 were generated by fusing the

ENO1 cDNA to the CaMV 35S promoter and transforming the construct

into Arabidopsis. PCR primers were designed for the full-length coding

sequence of ENO1 (At1g74030). A sense primer ENO1-GW-F (59-CAC-

CATGGCTTTGACTACAAAACC-39) containing the start codon, and an

antisense primer containing the stop codon ENO1-2GW-R (59-TCA-

TGGTGATCGGAAAGCTTCACC-39) was designed for cloning into the

Gateway entry vector pENTR D-TOPO. The PCRs were run at 958C for 2

min followed by 35 cycles (30 s at 958C, 30 s at 598C, and 1 min at 728C)

and a final extension at 728C for 7min. Full-length ENO1was recombined

by LR reaction into the Gateway destination vector pGWB2 and trans-

formed into DH10B competent cells. Transgenic plants were generated

by vacuum infiltration of Arabidopsis flowers using Agrobacterium

tumefaciens cultures containing the appropriate construct (Clough and

Bent, 1998). The primary transformants were allowed to flower and

produce seeds. Transformants were selected with kanamycin and ver-

ified by PCR analysis.

Microscopy and Histochemical Analyses

For all histochemical analyses, the Eclipse E800 microscope (Nikon)

equipped with differential interference contrast and fluorescence optics

was used. Images were captured using a 1-CCD color video camera (KY-

F1030; JVC) operated by the DISKUS software package (Technisches

Buero Hilgers).

Pollen viability was assessed using Alexander staining (Alexander,

1969). Pollen grains released from the anthers were directly transferred to

the staining solution and further analyzed by light microscopy. Male

gametophyte development was analyzed by DAPI staining (Park et al.,

1998) on mature, dehisced, and indehiscent pollen.

Phenolic compounds of mature pollen grains were visualized by fluores-

cencemicroscopy in thepresenceof afluorescenceenhancer.Pollengrains

were incubated for 15 to 30min in 0.25%diphenylborate-2-aminoethyl and

0.005% Triton X-100 and the fluorescence subsequently visualized with an

excitation of 330 nm < l < 380 nm and an emission filter l > 420 nm.

Lignification of cell walls was analyzed in cross-sections of inflores-

cence stems 1 mm above the rosette with ACF (i.e. equal portions of

astrablue, chrysoidin and neofuchsin solution at a concentration of

1 mg·mL21 each; Etzold, 2002). In the presence of ACF, lignin stains

red, suberin and cutin yellow, and cellulose cell walls are contrasted blue.

The cross-sections were immersed in the ACF staining solution and

immediately analyzed by light microscopy.

Developing ovules were analyzed by differential interference contrast

light microscopy. Ovules were removed from the pistil or the silique and

immersed in a droplet of water on a slide and covered with a cover slip.

The whole-mount ovules were examined.

TB staining of rosette leaves and stems was essentially done as

described by Tanaka et al. (2004).

Confocal Laser Scanning Microscopy

Phenolic compounds in anthers were analyzed by confocal laser scan-

ning microscopy (excitation, 488 nm; emission, 500 to 550 nm) with a

Leica SP2 microscope. Images were captured by the LCS software and

further processed using Adobe Photoshop 6.

Scanning Electron Microscopy

Cutical wax crystals on the epidermis of inflorescence stems were

visualized by scanning electron microscopy with a Leica Electron Optics

430i using the image analysis software SIS version 2.11. The stem

samples were placed untreated (i.e., not sputtered) directly into the

vacuum of the scanning electron microscopy device.

Ultrastructural Analysis

For transmission electronmicroscopy of pollen, stamenswere taken from

different flower stages and fixed with 2% glutaraldehyde in 50 mM

phosphate buffer, pH 7.2, overnight at 48C. Sampleswere postfixed in 1%

osmium tetroxide for 8 h on ice, dehydrated in a graduated acetone

series, including a step with 1% uranylacetate (in 50% acetone, 2 h),

2612 The Plant Cell

embedded in Spurr’s resin, and polymerized at 608C for 72 h. Ultrathin

sections (60 to 70 nm) were cut with a diamond knife (Diatome) on a Leica

EM UC6 ultramicrotome (Leica Microsystems) and mounted on pioloform-

coated copper grids. The sections were stained with lead citrate and

uranyl acetate (Reynolds, 1963) and viewed with a JEOL JEM-2100

transmission electron microscope operated at 80 kV. Micrographs were

taken using a 4k 3 4k Gatan UltraScan 4000 CCD camera. The sections

were also stained using TB stain (a 4:1 mixture of 1% TB, 1% borax, and

1% pyromin A) for light microscopy viewing.

In Vitro Germination of Pollen

The germination efficiency of pollen grains was assessed in vitro

according to Fan et al. (2001). For each experiment, freshly anther-

dehisced flowers were randomly collected from five different plants and

were dipped on agar plates to transfer the pollen grains to the germination

medium (5 mM MES/Tris, pH 5.8, 1 mM KCl, 10 mM CaCl2, 0.8 mM

MgSO4, 1.5 mM boric acid, 1% [w/v] agar, 16.6% [w/v] sucrose, 3.65%

[w/v] sorbitol, and 10 mg mL21 myo-inositol). For the germination assay,

plates were incubated for 20 h in a climate-controlled chamber at 228C

with 100%humidity in low light (30mmolm22 s21) supplied by fluorescent

tubes. For each agar plate, 100 pollen were counted and the germination

percentage calculated.

Chlorophyll Leaching Assay

Chlorophyll leaching from rosette leaves was determined as described by

Aharoni et al. (2004). Whole rosettes of 3-week-old plants were rinsed

with tap water, weighed, and transferred to tubes containing 30 mL of

80% ethanol. The rosettes were incubated under gently agitation in the

dark. For chlorophyll determination, 1 mL of the ethanol was removed

fromeach sample every 10min during the first hour and finally after 90 and

120 min. Chlorophyll contents were measured at 664 and 647 nm.

Amino Acid Analysis

For the analysis of free amino acid contents, the tissue (100 to 200mg) was

ground to a fine powder under liquid nitrogen and subsequently extracted

with 50mL of 1M perchloric followed by 50mL of 0.1Mperchloric acid. The

pH of the extracts was adjusted to 7.0 with 9.3 mL of 5 M potassium

carbonate. The mixture was centrifuged for 30 min at 20,800g at 48C. The

supernatants were transferred to fresh tubes for HPLC analysis. The

samples were precolumn derivatized with o-phthalaldehyde (Lindroth and

Mopper, 1979) and separated onaHewlett PackardHP1100HPLCsystem,

using a Nucleodur 100-5 C18 ec column (Macherey and Nagel). An

optimized stepwise gradient ranging from 7% buffer A (50% methanol

and 50% acetonitrile) and 93% buffer B (40 mM sodium acetate, pH 6.5) to

80% buffer A was used for elution at 358C for 33 min at a flow rate of

1 mL·min21 after injection of 5-mL sample. Derivatives were detected by

excitation at 230 nm and their fluorescence at 450 nm. Alternatively freshly

ground frozen plant tissue was extracted for 20min at 48C sequentially with

900 mL 80% (v/v), 300 mL 50% (v/v), and 300 mL 80% (v/v) aqueous

methanol (buffered with 2.5 mM HEPES/KOH, pH 7.5). Upon precolumn

derivatization with orthophtalaledehyde, 5 mL of the derivates were

subjected to HPLC analysis using a Hyperclone C18 BDS column (Phe-

nomenex) connected to an Ultimate 3000 HPLC system coupled with the

RF2000 fluorescence detector (Dionex). Fluorescent derivates were eluted

(1.1 mL·min21) at 358C by a nonlinear solvent gradient: 100% A (8.8 mM

sodium hydrogenphosphate, pH 7.5, and 0.2% tetrahydrofuran) for 0 to 2

min, 15% B (19 mM 8.8 mM sodium hydrogenphosphate, pH 7.5, 33%

methanol, and 21%acetonitrile) for 2 to 8min, 18%B for 8 to 10min, 50%B

for 10 to 14min, 52% B for 14 to 18min, 60% B for 18 to 25min, 100% for

25 to 26 min, 100% B for 26 to 28 min, and 0% B for 28 to 30 min.

Fatty Acid Analysis

Total fatty acids in seeds were quantified by gas chromatography after

derivatization to fatty acid methyl esters using pentadecanoic acid (15:0)

as internal standard (Browse et al., 1986). The desaturation index (ID) was

calculated as follows:

ID = (18:1 + 2·18:2 + 3·18:3 + 20:1 + 22:1)/(14:0 + 16:0 + 18:0 + 20:0 + 22:0)

Analysis of Cuticular Wax

Wax analysis was conducted as described in detail by Kurdyukov et al.

(2006). Arabidopsis shoots were immersed for 10 s in chloroform (CHCl3;

Merck) for wax extraction. An internal standard (20 mL of tetracosane

[Fluka]; chloroform solution 10mg into 50 mL) was added to each sample

directly after wax extraction. Hydroxyl and carboxylic groups were

transformed into corresponding trimethylsilyl derivatives by reaction

with N,N-bis-trimethylsilyl-trifluoroacetamide (Macherey-Nagal) in pyri-

dine (30 min at 708C). A sample of 1 mL was analyzed by gas chroma-

tography with flame detection (CG-Hewlett Packard 5890 series H;

Hewlett-Packard) with on-column injection (30 m DB-1 i.d. 0.32 mm,

film 0.2 mm; JandW Scientific). Wax compounds were identified by gas

chromatography–mass spectrometry (quadrupole mass selective detec-

tor HP 5971; Hewlett-Packard).

Determination of Phytohormones

The determination of the phytohormones IAA, ABA, oPDA, JA, SA, and

SAG was performed by liquid chromatography–tandem mass spectrom-

etry as previously described (Luo et al., 2009). For analysis of SAG, mass

transitions were as follows: 299/137 (declustering potential 245 V,

entrance potential 27 V, and collision energy 222 V).

For determination of the relative amount of zeatin, samples were

extracted using the method described by Matyash et al. (2008). The polar

phases were analyzed by an Ultra Performance LC (ACQUITY UPLC

system; Waters) combined with a time-of-flight mass spectrometer (LCT

Premier; Waters). For liquid chromatography, an ACQUITY UPLC BEH

SHIELD RP18 column (1 3 100 mm, 1.7-mm particle size; Waters) was

used at a temperature of 408C, a flow rate of 0.2 mL/min, and with the

following gradient: 0 to 0.5 min 100%A, 0.5 to 3min from 0%B to 20%B,

3 to 8 min from 20% B to 100% B, 8 to 10 min 100% B, and 10 to 14 min

100% A (solvent system A, water/methanol/acetonitrile/formic acid

[90:5:5:0.1, v/v/v/v]; B, acetonitrile/formic acid [100:0.1, v/v]).

The TOF-MS was operated in positive electrospray ionization mode in

W optics with a mass resolution larger than 10,000. Data were acquired

by MassLynx software in centroided format over a mass range of m/z 50

to 1200 with a scan duration of 0.5 s and an interscan delay of 0.1 s The

capillary and the cone voltage were maintained at 2700 and 30 V and the

desolvation and source temperature at 250 and 808C, respectively.

Nitrogen was used as cone (30 L/h) and desolvation gas (600 L/h). For

accurate mass measurement of > 5 ppm root mean squared, all analyses

were monitored using Leu-enkephaline ([M+H+] 556.2771 m/z; Sigma-

Aldrich) as lock spray reference compound at a concentration of 0.5 mg/

mL in acetonitrile/water (50:50, v/v) and a flow rate of 30 mL/min.

Determination of Flavonoids

For flavonoid measurements, six flowers were pulverized using liquid

nitrogen and extracted as described previously (Pinto et al., 1999) in 200

mL of methanol:water:HCl (70:20:1 by volume) at 608C for 1 h (99.8

methanol and 35.4%HCl), and the absorption at 310 nmwas determined

after cooling down the extracts to room temperature. As a standard,

naringinin (Sigma-Aldrich) was used.


Determination of Protein, Soluble Sugars, and Starch in Seeds

For total protein analysis, 10 seeds were homogenized in 100 mL of

extraction buffer (25 mM Tris-HCl, pH 8.0, 125 mM NaCl, 0.5 mM EDTA,

and 0.5% [w/v] SDS) (Ruuska et al., 2002). The total protein content was

measured in 100 mL of crude homogenate with a Bio-Rad detergent-

compatible protein assay system using g-globulin as standard according

to the manufacturer’s instructions. Starch and soluble sugars were

essentially extracted and determined enzymatically as described by Lin

et al. (1988) starting from 35 seeds per sample.

Photosynthesis Measurements

Modulated chlorophyll a fluorescence emission from the upper leaf

surface was measured with a pulse amplitude modulation fluorometer

(IMAGING PAM, Maxi version; Walz) according to the principles of

Schreiber et al. (1986). Photosynthetic ETRs were assessed according

to Genty et al. (1989).

Statistical Evaluation of Experimental Data

The data are expressed as mean values 6 SE of the indicated number of

independent measurements. Significant differences between two data

sets were analyzed using theWelch test, which allows for unequal relative

errors between two groups of measurements assuming that a Gauss

distribution is applicable (Welch, 1947). For data plotting, SigmaPlot8.0

for Windows (SPSS) was used.

Accession Numbers

Sequence data from this article can be found in the Arabidopsis Ge-

nome Initiative or GenBank/EMBL databases under the following

accession numbers: Actin2 (At5g09810; NM_121018), BDG (At1g64670;

NM_105142), CER1 (At1g02205; NM_180601), CER10 (At3g55360; NM_

115394), ENO1 (At1g74030; NM_106062), KCR1 (At1g67730; NM_105441),

KCR2 (At1g24470; NM_102292), PPT1 (At5g33320; NM_122856), PPT2

(At3g01550; NM_111021), andWAX2 (At5g57800; NM_125164).

Supplemental Data

The following materials are available in the online version of this article

Supplemental Figure 1. Comparison of the Shoot and Root Pheno-

types of Heterozygous eno1 Mutants in the Homozygous cue1

Background (ccEe) with cue1 Single Mutants.

Supplemental Figure 2. Confocal Microscopy Images of Pollen

Sacs from Col-0 Wild-Type Plants and Heterozygous ccEe Double

Mutants.

Supplemental Figure 3. Autofluorescence of Pollen from a Wild-Type

Plant and Heterozygous eno1 Mutants in the Homozygous cue1

Background (ccEe).

Supplemental Figure 4. Comparison of Pollen Germination Rates

between Arabidopsis Wild-Type (Col-0) and ccEe Plants with Alleles

cue1-1/eno1-2(+/2) and cue1-6/eno1-2(+/2).

Supplemental Figure 5. Relative Contents of Amino Acids Extracted

from Flower Buds or Rosette Leaves of the Wild Type (Col-0), cue1-6,

and eno1 Single Mutants as well as the Heterozygous eno1 Mutants

in the Homozygous cue1 Background (ccEe).

Supplemental Figure 6. Cross Sections of the Inflorescence Stem of

Heterozygous eno1 Mutants in the Homozygous cue1 Background

(ccEe) Compared with Wild-Type and cue1 Plants.

Supplemental Figure 7. Typical Toluidine Blue Staining for Cuticle

Integrity of Aerial Parts of the Heterozygous eno1 Mutant in the

Homozygous cue1 Background (ccEe) Compared with the Wild Type

(pOCA = Ecotype Bensheim, Col-0) and the cue1 and eno1 Single

Mutants.

Supplemental Figure 8. Scanning Electron Microscopy Images of

Epiculticular Wax Crystals of Inflorescence Stems of Arabidopsis Wild

Type (Col-0, pOCA = Ecotype Bensheim) as well as Mutant Alleles of

cue1, eno1, and ccEe.

Supplemental Figure 9. Epicuticular Wax Analysis of Inflorescence

Stems of Arabidopsis Wild Type (pOCA = Ecotype Bensheim, Col-0)

as well as Alleles of cue1, eno1, and cue1-1/eno1-2(+/2).

Supplemental Figure 10. Chl Leaching Experiment with Cut Leaves

of 6-Week-Old Col-0, eno1-2, cue1-6, and cue1-6/eno1-2(+/2).

Supplemental Figure 11. Light Microscopy Images Showing the

Distribution of Stomata as well as the Phenotypes of Stomatal Guard

Cells on the Adaxial Surface of Rosette Leaves of the Wild Type

(Col-0), the cue1-6 and eno1-2 Single Mutants, as well as in the

Heteozygous eno1-2 Mutant in the Homozygous cue1-6 Background

[cue1-6/eno1-2(+/2)].

Supplemental Figure 12. Relative Transcipt Levels of Genes In-

volved in Cuticular Wax Biosynthesis in cue1-6/eno1-2(+/2) Com-

pared with the Wild Type.

Supplemental Figure 13. Relative Composition of Saturated and

Unsaturated Fatty Acids Determined by Gas Chromatography after

Derivatization to Fatty Acid Methyl Esters.

Supplemental Figure 14. Spatial and Developmental Expression

Profiles of Genes Involved in PEP Delivery to Plastids.

Supplemental Table 1. Genotype Analysis of the F2 Generation of

Crosses between cue1 and eno1 Mutants.

Supplemental Table 2. Content of Flavonoids in Flowers of Arabi-

dopsis Wild-Type Plants, cue1 and eno1 Mutant Alleles, and Hetero-

zygous eno1-2 Mutants in the Homozygous cue1-6 Background.

Supplemental Table 3. Detailed Tissue and Development-Specific

Expression Profiles of Genes Involved in PEP Provision to Plastids as

well as Pyruvate Synthesis in Plastids and the Cytosol.

Supplemental Table 4. Primer Pairs Used for qRT-PCR.

ACKNOWLEDGMENTS

We thank the Deutsche Forschungsgemeinschaft and the International

Graduate School in Genetics and Functional Genomics (Cologne) for

funding. We thank Tanja Fuchs (University of Bonn) for help with fatty

acid measurements. We also thank K.H. Linne von Berg (University of

Cologne) for help with the scanning electron microscopy and Mojgan

Shahriari (University of Cologne) for the introduction into confocal laser

scanning microscopy.

Received November 28, 2009; revised July 1, 2010; accepted August 4,

2010; published August 26, 2010.

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DOI 10.1105/tpc.109.073171; originally published online August 26, 2010; 2010;22;2594-2617Plant Cell

Ulf-Ingo Flügge and Rainer E. HäuslerLukas Schreiber, Cornelia Göbel, Kirstin Feussner, Ivo Feussner, Kay Marin, Pia Staehr, Kirsten Bell,

Veena Prabhakar, Tanja Löttgert, Stefan Geimer, Peter Dörmann, Stephan Krüger, Vinod Vijayakumar,Arabidopsis thalianaDevelopment in

Phosphoenolpyruvate Provision to Plastids Is Essential for Gametophyte and Sporophyte

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Phosphoenolpyruvate Provision to Plastids Is Essential for ...Phosphoenolpyruvate Provision to Plastids Is Essential for Gametophyte and Sporophyte Development in Arabidopsis thaliana